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.

Cathepsins in Rotator Cuff Tendinopathy: Identification in Human Chronic Tears and Temporal Induction in a Rat Model.

While overuse of the supraspinatus tendon is a leading factor in rotator cuff injury, the underlying biochemical changes have not been fully elucidated. In this study, torn human rotator cuff (supraspinatus) tendon tissue was analyzed for the presence of active cathepsin proteases with multiplex cysteine cathepsin zymography. In addition, an overuse injury to supraspinatus tendons was induced through downhill running in an established rat model. Histological analysis demonstrated that structural damage occurred by 8 weeks of overuse compared to control rats in the region of tendon insertion into bone. In both 4- and 8-week overuse groups, via zymography, there was approximately a 180% increase in cathepsin L activity at the insertion region compared to the controls, while no difference was found in the midsubstance area. Additionally, an over 400% increase in cathepsin K activity was observed for the insertion region of the 4-week overused tendons. More cathepsin K and L immunostaining was observed at the insertion region of the overuse groups compared to controls. These results provide important information on a yet unexplored mechanism for tendon degeneration that may operate alone or in conjunction with other proteases to contribute to chronic tendinopathy.

The effect of diclofenac on matrix metalloproteinase levels in the rotator cuff.

INTRODUCTION: Matrix metalloproteinases (MMPs) are involved in physiological events such as restructuring of the tissue, morphogenesis, wound healing and normal developmental process. Use of diclofenac sodium following rotator cuff repair can disrupt healing of tendon through acting on MMPs. MATERIALS AND METHODS: Supraspinatus tendons of rats (n = 84) were detached from their insertion on humerus, and repaired to anatomic footprint. Rats were divided into study group (n = 42) and control group (n = 42). Study group received a dose of 1 mg/kg daily diclofenac sodium subcutaneously. The rats were killed at weeks 1, 3 and 6, and seven rats from each groups were included in biomechanical and immunohistological examinations. Immunohistological staining of MMP-2, MMP-3 and MMP13 were used. RESULTS: Maximum load was reduced in the study group at the end of week 1 (8.76 vs. 5.28 N) (p = 0.01). MMP-3 level was statistically significantly lower in the study group at the end of week 1. MMP-13 level and stiffness decreased towards week 6 in the study group while in the control group the level of MMP-2 decreased towards week 6. CONCLUSION: Diclofenac has an impact on the levels of MMP-2, MMP-3 and MMP-13, which are needed for normal healing process, and it can also lead to disruption of tendon healing.

Apoptosis occurs throughout the diseased rotator cuff.

BACKGROUND: Even though apoptosis is known to be closely associated with rotator cuff tears, the differences in apoptosis according to the location within the torn supraspinatus tendon are still unknown. PURPOSE: To elucidate where apoptosis begins within the supraspinatus tendon. STUDY DESIGN: Controlled laboratory study. METHODS: Tendon tissues were collected from 14 patients undergoing arthroscopic rotator cuff repair surgery and 7 patients undergoing surgery for proximal humeral fracture who served as controls. In the patients with rotator cuff tears, the samples were harvested at 3 sites: the most lateral torn margin, 1 cm medial from the torn margin, and at the posterior torn corner. Caspase 3/7, 8, and 9 and cytochrome c activities were measured to determine the intracellular apoptosis pathway. Apoptotic cells were determined by in situ TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining, and immunohistochemistry was performed. RESULTS: The apoptotic activities of tendons from the experimental subjects were significantly higher than those of the controls. There were, however, no significant differences between the 3 sample sites. Immunohistochemistry also revealed strong expression of increased caspase 3/7, 8, and 9 and cytochrome c but no significant difference between them. CONCLUSION: This study shows that the intracellular apoptotic pathway is not only through the cell membrane receptor but also via intracellular mitochondria cascade. CLINICAL RELEVANCE: Because apoptosis occurs regardless of the location within the rotator cuff, debridement of the torn margin to obtain a healthy tendon may not be needed. Further study should focus on not only the technique of tying the torn tendon back to the bone but also biological augmentation to reverse or prevent further apoptosis within rotator cuff tendon.

Metalloproteases and their inhibitors are altered in both torn and intact rotator cuff tendons.

BACKGROUND: We evaluated the role of matrix metalloproteases (MMPs) and their inhibitors which are involved in extracellular matrix remodeling and degradation, in the pathogenesis of chronic rotator cuff tears. MATERIALS AND METHODS: Tendon samples were harvested from 13 patients who underwent arthroscopic repair of a rotator cuff tear. Supraspinatus biopsy specimens were harvested en bloc from the arthroscopically intact middle portion of the tendon more than 1 cm from the torn edge, from the lateral edge of the tear, and from the superior one third of the macroscopically intact subscapularis tendon used as control. Histological analysis and an evaluation of the activity of specific metalloproteases and the tissue inhibitors of metalloprotease (TIMP-1, TIMP-2) was done blindly by multiplex sandwich ELISA (Search-Light technology) in each specimen RESULTS: Histological evidence of tendinopathy was present in all patients with a tear of the rotator cuff, and not in the macroscopically intact subscapularis tendon. There was a significant increase in MMP 1, MMP 2, MMP 3 and in TIMP-1, TIMP-2 levels in all specimens examined, including the macroscopically intact portion of the supraspinatus tendon and in the control specimens CONCLUSIONS: The tissue in the ruptured area of the supraspinatus tendon undergoes marked rearrangement at molecular levels. This involves the activity of MMP 1, 2 and 3, and supports the critical role of MMPs in the tendon physiology. Seemingly intact parts of the injured supraspinatus tendon can present tendinopathic features, with altered cellular metabolism.

Platelet-rich plasma protects rotator cuff-derived cells from the deleterious effects of triamcinolone acetonide.

Triamcinolone acetonide (TA) injections are widely used to treat enthesopathy, but they may induce adverse effects such as tendon impairment and rupture. Platelet-rich plasma (PRP) is a blood fraction containing high platelet concentrations and various growth factors that play a role in tissue repair processes. The purpose of this study is to investigate whether TA has deleterious effects on human rotator cuff-derived cells, and if PRP can protect these cells from the effects of TA. Human rotator cuff-derived cells were cultured with and without TA and PRP, and the culture without any additive served as the control. Cell morphology was assessed at days 7 and 21. Cell viability was evaluated at days 1, 7, 14, and 21 by a water-soluble tetrazolium salt assay. Induction of apoptosis was measured by immunofluorescence staining and flow cytometry at day 7. Induction of cleaved caspase-3 was measured by immunofluorescence staining at day 7. The cells cultured with TA had a flattened and polygonal shape at day 7. The cells cultured with both TA and PRP were similar in appearance to control cells. Exposure to TA also significantly decreased cell viability, but cell viability did not decrease when PRP was added along with TA. The number of apoptotic cells increased with TA exposure, while addition of PRP prevented cell apoptosis. In conclusion, the deleterious effect of TA was prevented by PRP, which can be used as a protective agent for patients receiving local TA injections.

Role of metalloproteinases in rotator cuff tear.

The role of matrix metalloproteinases (MMPs) and their inhibitors (TIMPS) in the pathophysiology of rotator cuff tears has not been established yet. Recent advances empathize about the role of MMPs and TIMPS in extracellular matrix (ECM) remodeling and degradation in rotator cuff tears pathogenesis and healing after surgical repair. An increase in MMPs synthesis and the resulting MMPs mediated alterations in the ECM of tendons have been implicated in the etiopathogenesis of tendinopathy, and there is an increase in the expression of MMPs and a decrease in TIMP messenger ribonucleic acid expression in tenocytes from degenerative or ruptured tendons. Importantly, MMPs are amenable to inhibition by cheap, safe, and widely available drugs such as the tetracycline antibiotics and bisphosphonates. A better understanding of relationship and activity of these molecules could provide better strategies to optimize outcomes of rotator cuff therapy.

Expression of matrix metalloproteinases 1, 3, and 9 in differing extents of tendon retraction in the torn rotator cuff.

PURPOSE: Differing extents of tendon retraction are found in full-thickness rotator cuff tears. The pathophysiologic context of tendon degeneration and the extent of tendon retraction are unclear. Tendon integrity depends on the extracellular matrix, which is regulated by matrix metalloproteinases (MMP). It is unknown which enzymes play a role in tendon degeneration. The hypotheses are that (1) the expression of MMPs 1, 3, and 9 is altered in the torn rotator cuff when compared with healthy tendon samples; and (2) that there is a relationship between MMP expression and the extent of tendon retraction in the torn cuff. METHODS: Rotator cuff tendon samples of 33 patients with full-thickness rotator cuff tears (Bateman grade III) were harvested during reconstructive surgery. Samples were dehydrated and paraffin-embedded. Immunohistologic determination of MMP 1, 3, and 9 expression was performed by staining sample slices with MMP antibody. The extent of tendon retraction was determined intraoperatively according to Patte's classification and patients were assigned to 4 groups (control group, and by tendon retraction grade Patte I-III). The control group consisted of six healthy tendon samples. RESULTS: Expression of MMPs 1 and 9 was significantly higher in torn cuff samples than in healthy tendons whereas MMP 3 expression was significantly decreased (P < 0.05). MMP 9 expression significantly increased with rising extent of tendon retraction in the torn cuff (P < 0.05). No significant association was found between expression of MMPs 1 and 3 and the rising extent of tendon retraction by Patte's classification. CONCLUSION: Elevated expression of MMPs 1 and 9 as well as decreased MMP 3 expression can be detected in torn rotator cuff tendon tissue. There is a significant association between the extent of tendon retraction and MMP 9 expression. The results of this study give evidence that early surgical treatment of small and partial-thickness rotator cuff tears is required.

[Biochemical analysis of the synovial fluid of the shoulder joint in patients with and without rotator cuff tears]. / Biochemische Analyse der Synovialflüssigkeit bei Patienten mit und ohne Rotatorenmanschettendefekt.

AIM: The biochemical changes associated with rotator cuff tearing are still unclear. The aim of this study is to assess whether concentrations of matrix metalloproteinase in the synovial fluid are specifically altered in shoulders with torn rotator cuff tendons. PATIENTS AND METHOD: Synovial fluid was extracted via arthroscopy in 21 patients with complete rotator cuff tears (RCTs). The control group was composed of 21 patients without complete tears. The catabolic cartilage metabolism markers MMP-1 (collagenase), MMP-3 (stromelysin1) and MMP-13 (collagenase3) were quantified by an ELISA test and these results were then statistically analysed using SAS. RESULTS: The mean concentration of the 21 samples with rotator cuff tears shows a higher concentration of MMP 3 (2601.73 ng/mL vs. 1775.67 ng/mL) and MMP 13 (2.69 ng/mL vs. 2.35 ng/mL) as well as a significantly higher concentration of MMP 1 (p=0.0047) in the control group. CONCLUSIONS: A significant variation in the concentration of catabolic cartilage enzymes in the synovial fluid in patients with and without rotator cuff tears could not be found. Nonetheless, there is a bias for the MMP-3 and MMP-13 values, which makes a conductive influence in the aetiology of osteoarthritis probable.

Transglutaminases expression in human supraspinatus tendon ruptures and in mouse tendons.

The ethiopathogenesis of rotator cuff disease remains poorly understood. Many studies advocate the importance of extra cellular matrix for the homeostasis of connective tissue. Transglutaminase enzymes family has been studied in the context of connective tissue formation and stabilisation. Here, we investigated transglutaminases expression pattern in biopsies of normal and injured supraspinatus tendons of human shoulders and in the Achilles tendons of transglutaminase 2 knock-out and wild-type mice. Our results show that different transglutaminase family members are differentially expressed in human and mouse tendons, and that transglutaminase 2 is down-regulated at mRNA and protein levels upon human supraspinatus tendon ruptures.

Overexpression of nitric oxide synthases in tendon overuse.

Tendon disorders with a chronic nature, including the rotator cuff, are extremely common, and represent a major clinical problem. Mechanical overload has been proposed as an important etiologic factor in tendinopathies. Nitric oxide (NO), a free radical produced by nitric oxide synthases (NOSs), is a potent regulator and stimulator of biological processes including tendon degeneration and healing. It is also involved in response to mechanical stimuli in different tissues. In an animal model of acutely injured tendon healing temporal and differential expression of NOS isoforms has been demonstrated, suggesting that different patterns of NOSs expression may have different biological functions. Therefore, we hypothesized that tendon overuse may result in a differential upregulation of NOSs, particularly iNOS. An animal model of supraspinatus tendon overuse was utilized, which consisted of treadmill running. A group of animals of the same strain and age subjected to normal cage activity were used as controls. Following a 4-week exercise protocol supraspinatus tendons were harvested, RNA was extracted, and subjected to competitive reverse transcription and polymerase chain reaction (RT-PCR) to determine the expression levels of inducible-, endothelial-, and neuronal-NOS isoforms (i-, e-, and nNOS). The mRNA expression of all three NOS isoforms increased in the supraspinatus tendons as a result of overuse exercise. iNOS and eNOS mRNA expression increased fourfold (p < 0.01), and there was an increase, but statistically not significant, in nNOS mRNA expression in the overused tendons when compared with the controls. This study is the first to show that NOS isoforms are upregulated in rotator cuff tendon as a result of chronic overuse, and suggests the involvement of nitric oxide in the response of tendon tissue to increased mechanical stress.

Nitric oxide enhances collagen synthesis in cultured human tendon cells.

Collagen deposition is an important process that occurs during wound healing. We and others have shown that nitric oxide (NO) is important in tendon healing. The mechanisms whereby healing is enhanced are, however, undetermined. The aim of this study was to investigate whether NO could enhance collagen synthesis in cultured human tendon cells via exogenous NO and via an adenovirus containing the gene for inducible nitric oxide synthase (Ad-iNOS). Tendon cells from the torn edge of the tendons of patients undergoing rotator cuff repair surgery were cultured following collagenase digestion, and stimulated with exogenous NO (SNAP), transfected with Ad-iNOS, and treated with the NOS inhibitor, L-NMMA. Total protein and collagen synthesis were evaluated by (3)H-proline and collagenase sensitive (3)H-proline incorporation in human tendon cells. High doses of exogenous NO (SNAP) inhibited collagen synthesis. Lower doses enhanced total protein and collagen synthesis of the tendon cells. Ad-iNOS successfully transfected active iNOS into human tendon cells in vitro and also enhanced total protein and collagen synthesis of the tendon cells. The NOS inhibitor, L-NMMA, inhibited the effects of iNOS on the cells. Our studies show for first time that nitric oxide can enhance collagen synthesis in human tendon cells in vitro. These results may explain, in part, at least, the beneficial effects of NO donors in animal models and during the treatment of tendonopathies in human clinical trials. .

Expression and enzymatic activity of MMP-2 during healing process of the acute supraspinatus tendon tear in rabbits.

We investigated the spontaneous healing process of a surgically created supraspinatus tendon tear in rabbits with specific reference to the expression of matrix metalloproteinase-2 (MMP-2) and its time-course change in enzymatic activity along with the expression of tissue inhibitors of metalloproteinases (TIMPs). A transverse, full thickness tear of the supraspinatus tendon was created and examined. Immunohistochemical analysis revealed that MMP-2 positive cells were mainly localized at both cutting ends of the tendon, and reparative tissue encroached into the gap from the bursal side. The expression of TIMP-1 was induced in the cells at not only the tendon edges but also the reparative tissue during the healing process. TIMP-2 was constitutively expressed in both the tendon and the reparative tissue. Gelatin zymography using tissue culture media demonstrated latent and active forms of MMP-2 and characteristic time-linked changes of the enzymatic activity. Western blotting confirmed the bands as the latent form of MMP-2. These results suggest that MMP-2 is expressed and activated during the healing process of acute supraspinatus tendon tear and can play an important role in the remodeling process.

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.

Cell and matrix changes associated with pathological calcification of the human rotator cuff tendons.

The causative mechanism of tendon calcification ('calcifying tendinitis') is unknown. In this report, pathological human tendon samples were examined to give morphological and ultrastructural detail of the calcified regions and these findings were compared with those from normal tendon. Selected specimens were cryosectioned to enable histochemical and immunohistochemical comparison of the occurrence and distribution of specific matrix molecules in diseased and normal tendon tissues. The lack of collagen type II and alkaline phosphatase in the pathological regions suggests that the calcification process is not mediated through an endochondral transition. In contrast, the pathological areas were characterised by widespread labelling for chondroitin-4-sulphate/dermatan sulphate and intense pericellular localisation of chondroitin-6-sulphate.