Poster 100: Evaluation of SS-31 as a Potential Therapeutic in the Treatment of Tendinopathy
Authors: Szeto Hazel , Zhang Ying , Liu Shaohua , Rodeo Scott , ZHANG XUEYING 
Published: July 2022

Objectives:

The exact mechanism of tendinopathy is unknown and there are currently limited strategies to improve tendon healing. It is known that mitochondria have an important role in the development of tissue pathology. Previous work from our lab has demonstrated mitochondrial dysfunction in degenerative tendons from patients and from a shoulder impingement murine model. SS-31 is a mitochondrial-targeted antioxidant that targets the inner mitochondria membrane. It has been proven to be effective in numerous preclinical disease models and some are currently in Phase II clinical trials. The ability of SS-31 to target the mitochondria makes SS-31 a potential therapy for tendinopathy. The objective of this study is to investigate the therapeutic potential of SS-31 for the treatment of tendinopathy in a murine model and in human tenocytes.

Methods:

This study was approved by Weill Cornell IACUC and HSS IRB. In-vivo: Supraspinatus tendinopathy was induced by inserting a microsurgical clip in the mice subacromial space. The mice were divided into five groups (16/group): I: 4 weeks of impingement; II: 8 weeks of impingement; III: 8 weeks of impingement with 4 weeks SS-31 treatment (5 mg/kg/d); IV: 4 weeks of impingement, clip removal, then harvested 4 weeks later; V: 4 weeks SS-31 treatment after clip removal at 4 weeks impingement. Another 16 mice were used as controls. (Fig 1) Specimens were prepared for the following outcomes: (1) Biomechanical test; (2) Histological analysis. (3) Transmission electron microscopy (TEM). (4) Superoxidative dismutase (SOD) activity. In-vitro: tenocytes were isolated from tendon biopsies (Fig 6 and 7) and were then assigned to 4 groups (9/group): (1) healthy (n=9): tenocytes from non-injured tendons obtained from patients undergoing ACL reconstruction; (2) healthy with treatment: healthy tenocytes treated with 1μM SS-31 treatment for 72 hours; 3) degenerative: cells from degenerative supraspinatus tendons obtained patients undergoing rotator cuff repair; 4) degenerative with treatment: degenerative cells treated with 1μM SS-31 for 72 hours. All specimens were prepared for the following outcomes: (1) measurements of mitochondrial metabolism (SOD activity); (2) analysis of mitochondrial morphology (TEM); (3) membrane polarity assay using mito-trackergreen/Tetramethylrhodamine methyl ester staining); (4) cell viability (CCK8).

Statistical analysis:

One-way ANOVA with Tukey’s test were used. The significance limit was set at P<0.05.

Results:

In-vivo: Biomechanical test (Fig 2) revealed 61% and 66% decrease in failure force at 4 and 8 weeks compared to the intact tendon. The failure force gradually increased at 4 weeks after clip removal, after SS-31 treatment and when SS-31 treatment was combined with clip removal. The stiffness showed similar results. Histological analysis (Fig 3) demonstrated higher modified Bonar scores in the impingement groups; however, the tendon morphology tended to recover with a gradual improvement in Bonar score at 4 weeks following treatment, especially in the combined group. TEM images (Fig 4) demonstrated a decreased mitochondrial number with altered cristae organization and crista density in the impingement groups. With SS-31 and/or clip removal treatment, the structure and number of mitochondria normalized with improvement in cristae morphology. SOD activity (Fig 5) decreased after 4 and 8 weeks clip impingement compared to the control group and increased significantly following clip removal, with SS-31 treatment, and especially in the combined treatment group. In-vitro: SOD activity (Fig 8) was significantly lower in the degenerative group compared to the healthy group. SOD activity was upregulated in degenerative tenocytes after SS-31 treatment. TEM (Fig 9) images revealed no obvious change in mitochondria morphology and cristae organization after SS-31 treatment in healthy tenocytes. In contrast, disorganization and reduced density of the cristae with changes in ultrastructural integrity were observed in degenerative tenocytes. There were more organized cristae after SS-31 treatment in degenerative tenocytes, with some degree of recovery after SS-31 treatment. There was a significant decrease in the mitochondria number per tenocyte in the degenerative group. After the treatment, the number of mitochondria increased but did not reach the level seen in normal tenocytes. The fraction of cells with depolarized mitochondria (Fig 10) was increased in the degenerative group compared to healthy tenocytes, with a decrease following SS-31 peptide treatment for 72 hours. Cell viability (Fig 11) of the degenerative tenocytes was decreased (33.33 ± 5.03%) compared to the healthy tenocytes, with an increase to 50.44 ± 13.03% following treatment with SS-31.

Conclusions:

The study demonstrates the potential for SS-31 to treat mitochondrial dysfunction in tendinopathy and to aid in tendon healing, based on both in vivo (animal model) and in vitro (human tenocytes) experiments. Tendinopathic changes were confirmed in both the mouse model and isolated tenocytes from human specimens. Supraspinatus tendon structure and cell viability improved following clip removal and with SS-31 treatment, especially when the two strategies were combined. The clip removal may mimic the effect of acromioplasty that is clinically used in the treatment of impingement and SS-31 treatment could possibly be considered a conservative strategy. Mitochondrial dysfunction was observed in the development of tendinopathy and was reversed by SS-31 treatment and/or clip removal, with the most effective outcome in the combined group. These data demonstrate that SS-31, as a mitochondrial protectant, may be a useful strategy to promote healing of tendinopathy and might be most effective when combined with surgical treatment. [Figure: see text][Figure: see text][Figure: see text][Figure: see text][Figure: see text][Figure: see text][Figure: see text][Figure: see text][Figure: see text][Figure: see text][Figure: see text]