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Seventy-eight Sprague-Dawley rats (400-450 g) were used in this study. These rats were divided into 4 groups, and within each group were sacrificed at both 4 and 8 weeks after tendon detachments. The treatment groups consisted of: uninjured control (n=10 at 4 weeks, n=10 at 8 weeks), supraspinatus tendon detachment (n=10 at 4 weeks, n=10 at 8 weeks), supraspinatus+infraspinatus tendon detachments (n=10 at 4 weeks, n=9 at 8 weeks), and supraspinatus+subscapularis tendon detachments (n=9 at 4 weeks, n=10 at 8 weeks). In the tendon detachment groups, a unilateral surgery was performed on the

animal’s left shoulder to detach the prescribed rotator cuff tendons from their bony insertion on the humerus. The method for supraspinatus tendon detachment is well- established and has been used in this lab extensively.4,6-8,14,15,18 This technique has been recently expanded to include additional detachments of the other major rotator cuff tendons, the infraspinatus and subscapularis.4,14

The surgical detachment begins with a 2-cm skin incision made over the

glenohumeral joint with the arm in external rotation. This incision was then followed by blunt dissection through the deltoid muscle down to the rotator cuff musculature. The rotator cuff was then exposed and the tendons and their insertions onto the humerus were visualized. The tendons were then identified as: the subscapularis, the most anterior and broadest of the rotator cuff tendons; the supraspinatus, which passes under the bony arch of the acromion, coracoid and clavicle; and the infraspinatus, posterior to the others with an insertion similar to the supraspinatus. A traction suture was then passed underneath the acromion to facilitate further exposure of the tendons. The supraspinatus tendon was then separated from the other rotator cuff tendons anteriorly and posteriorly before sharply detaching it with a scalpel blade from its insertion on the greater tuberosity. In the two-tendon detachment groups, the other tendon (infraspinatus or subscapularis) was detached in the same manner. Any remaining fibrocartilage was left intact at the

insertion site and detached tendons were allowed to freely retract. The overlying muscle was then closed with suture and the overlying skin with staples. The rats were allowed unrestricted cage activity until sacrifice at 4 or 8 weeks.

After sacrifice, the scapula-biceps tendon-muscle segments were dissected out. The associated muscle was removed by gentle scraping and the tendon was fine dissected

under a stereomicroscope. In order to optically determine the distribution of strain along the tendon, Verhoeff stain lines were then placed along the tendon length using 6-0 silk suture. The first stain line was placed at the insertion of the tendon onto the scapula, and the remaining stain lines were placed 1.5, 3.5, 8.5 and 11.5 mm distally from the insertion in order to denote the proximal tendon insertion site (0-1.5 mm), intra-articular space (1.5-3.5 mm), and portion of the tendon in the bicipital groove (3.5-8.5 mm) (Figure 2.4). The last stain line was placed at 11.5 mm, denoting placement of the grip for mechanical testing. These positions were determined from a preliminary study consisting of

dissection of two rats, where the length and position of the tendon in the bicipital groove was determined with the animals arm at its side in neutral rotation. Two additional shoulders were fixed, decalcified, embedded, sectioned and stained with hematoxylin and

A

B

C

A

B

C

Figure 2.4: Image obtained during biomechanical testing illustrating regions of biceps tendon denoted by stain lines.

(A) insertion site; (B) intra-

articular space; (C) bicipital groove.

eosin. These sections were then examined under a microscope and the length of the tendon cross-sectional area for each location. After this measurement, the scapula was embedded in polymethylmethacrylate for biomechanical testing. The embedded scapula was placed into a custom designed testing fixture and the proximal end of the tendon (stain line at 11.5 mm) was held in a screw clamp lined with fine grit sandpaper. The specimen was then immersed in a 39ºC PBS bath and preloaded to 0.1 N before

preconditioning for 10 cycles from 0.1 to 0.5N at a rate of 1%/second. The tendon was then held for 300 seconds before a stress-relaxation experiment that elongated the

specimen to 4% strain at 5%/second (0.575 mm/sec) followed by a 600 second relaxation period. Following this relaxation period, the specimen was returned to its initial preload displacement and held for 60 seconds before ramp to failure at 0.3%/second was applied. This mechanical testing procedure can be seen in Figure 2.5.

Local tissue strain in each portion of the tendon was measured optically using the applied stain lines and a custom MATLAB program. Briefly, this program determines the difference in gray scale intensity between the stain line and the surrounding tendon through the images collected during the ramp to failure test. Elastic properties, such as stiffness and modulus, were calculated using the linear portion of the load-displacement and stress-strain curves, respectively. Maximum stress at the insertion was calculated only for specimens that failed at the insertion site and not for specimens that failed at any other location, such as the grip or midsubstance. Viscoelastic properties (peak and equilibrium load, percent relaxation) were determined from the stress relaxation curve for each specimen.

Significance was assessed between groups at each time point using one-way ANOVAs with Bonferroni post-hoc correction. After it was determined that control tendons exhibited a difference between time points, a bootstrapping approach was used to randomly pair 4 and 8 week data from each group.17 The difference in each property over time (from 4 to 8 weeks) was then compared between groups using a one-way ANOVA and Bonferroni correction. To correct for the number of comparisons, significance was set at p<0.0125 (0.05/4) and trends at p<0.025 (0.1/4).