Full-thickness rotator cuff tear in rat results in distinct temporal expression of multiple proteases in tendon, muscle, and cartilage.

The etiology of joint tissue degeneration following rotator cuff tear remains unclear. Thus, the purpose of this study was to understand the timeline of protease activity in the soft tissues of the shoulder (tendon, muscle, and cartilage) that may lead to down-stream degeneration following rotator cuff tear. A well-established rat model involving suprascapular nerve denervation and supraspinatus/infraspinatus tendon transection was employed. Histological staining and/or micro-computed tomography (µCT) were used to observe structural damage in the supraspinatus tendon and muscle, humeral head cartilage, and subchondral bone. Multiplex gelatin zymography was utilized to assess protease activity in the supraspinatus tendon and muscle, and humeral head cartilage. Zymography analysis demonstrated that cathepsins were upregulated in the first week in all tissues, while MMP-2 maintained prolonged activity in supraspinatus tendon between 1 and 3 weeks and increased only at 3 weeks in supraspinatus muscle. In supraspinatus tendon, increased cathepsin L and MMP-2 activity in the first week was concurrent with matrix disorganization and infiltration of inflammatory cells. In contrast, significant upregulation of cathepsin L and K activity in supraspinatus muscle and humeral head cartilage did not correspond to any visible tissue damage at 1 week. However, focal defects developed in half of all animals' humeral head cartilage by 12 weeks (volume: 0.12 ± 0.09 mm3 ). This work provides a more comprehensive understanding of biochemical changes to joint tissue over time following rotator cuff tear. Overall, this provides insight into potential therapeutic targets and will better inform ideal intervention times and treatments for each tissue. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:490-502, 2019.

Supraspinatus tendon overuse results in degenerative changes to tendon insertion region and adjacent humeral cartilage in a rat model.

The etiology of rotator cuff tendon overuse injuries is still not well understood. Furthermore, how this overuse injury impacts other components of the glenohumeral joint, including nearby articular cartilage, is also unclear. Therefore, this study sought to better understand the time course of tendon protease activity in a rat model of supraspinatus overuse, as well as determine effects of 10 weeks of overuse on humeral head articular cartilage. For these studies, multiplex gelatin zymography was used to characterize protease activity profiles in tendon and cartilage, while histological scoring/mechanical testing and micro-computed tomography (µCT) imaging were used to quantify structural damage in the supraspinatus tendon insertion and humeral articular cartilage, respectively. Histological scoring of supraspinatus tendon insertions revealed tendinopathic cellular and collagen fiber changes after 10 weeks of overuse when compared to controls, while mechanical testing revealed no significant differences between tensile moduli (overuse: 24.5 ± 11.5 MPa; control: 16.3 ± 8.7 MPa). EPIC-µCT imaging on humeral articular cartilage demonstrated significant cartilage thinning (overuse: 119.6 ± 6.34 µm; control: 195.4 ± 13.4µm), decreased proteoglycan content (overuse: 2.1 ± 0.18 cm-1 ; control: 1.65 ± 0.14 cm-1 ), and increased subchondral bone thickness (overuse: 216.2 ± 10.9 µm; control: 192 ± 17.8µm) in the overuse animals. Zymography results showed no significant upregulation of cathepsins or matrix metalloproteinases in tendon or cartilage at 2 or 10 weeks of overuse compared to controls. These results have further elucidated timing of protease activity over 10 weeks and suggest that damage occurs to other tissues in addition to the supraspinatus tendon in this overuse injury model. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1910-1918, 2017.

Human shoulder tendon biopsy samples in organ culture produce procollagenase and tissue inhibitor of metalloproteinases.

OBJECTIVE: To investigate the production of the matrix metalloproteinase (MMP), collagenase (MMP-1), and its natural inhibitor, the tissue inhibitor of metalloproteinases (TIMP) by diseased human tendon samples in organ culture. METHODS: Portions of tendons were excised from the shoulders of patients undergoing shoulder surgery, classified as either proximal to the lesion (abnormal) or distal to the lesion (normal) according to their macroscopic appearance at surgery, and placed in organ culture for periods of up to 28 days. The release of collagenase and TIMP activity in the conditioned culture medium was measured. RESULTS: Procollagenase and TIMP were both produced by all the tendon samples for an extended period of time. The levels of enzyme and inhibitor varied between patients, but in most of them TIMP levels were greater than collagenase levels. In one sample of calcified tendon, procollagenase levels were greater than those of TIMP. The mean level of collagenase produced by tendon proximal to the lesion and tendon distal to the lesion were not significantly different (95.2 (SD 106.8) U/g and 34.0 (45.3) U/g, respectively), while the corresponding figures for TIMP were 109.7 (62.3) U/g and 53.0 (27.9) U/g (p = < 0.05), although there was considerable variation in some samples. Western blotting and collagen fragment analysis confirmed that the collagenolytic activity detected was attributable to the metalloproteinase fibroblast collagenase (MMP-1). CONCLUSIONS: Tendon tissue can actively secrete procollagenase, an enzyme that, once activated, is capable of remodelling collagen, the major connective tissue component of tendon. Collagenase is produced even in unstimulated cultures, although the concentrations of TIMP are usually greater than that of collagenase in most samples. Some activation of collagenase appeared to have occurred. These results indicate that tendon tissue cells are capable of producing a remodelling response, even in end stage tendon disease.

Effects of exercise on the histochemical changes of articular chondrocytes in adult dogs.

Effects of exercise regimens on the enzyme histochemical changes of articular chondrocytes of the humeral heads in adult shepherd-type dogs were studied. One group of 4 dogs was exercised by walking on a flat surface 5 days a week for 6 months. A 2nd group of 4 dogs was exercised under the same conditions, except that the dogs were forced to walk over platforms placed in their path. Three control dogs were exercised ad libitum in their housing area. In all dogs, the reactivity of lactic acid dehydrogenase was quite strong nicotinamide dinucleotide dehydrogenase was moderate, and glucose-6-phosphatase was week. Succinic acid dehydrogenase uridine diphosphate (UDP)-galactose-4-epimerase, and UDP-N-acetylglucosamine-4-epimerase were of weakly moderate staining reactivity. Consistent regional or laminar variability was not found among the chondrocytic populations of the exercised and control groups for the reactivity of the enzymes studied. However, regional and/or laminar variabilities in individuals of the experimental groups were identified. The weak reactivity of glucose-6-phosphatase as seemingly contradictory to the presence of intracellular lipids of adult articular chondrocytes. Lipid synthesis was suggested as a mechanism to store excessive quantities of hydrogen ions in an innocuous form, rather than in the potentially deleterious by-product of anaerobic glycolysis, lactic acid.