Adipose Stem Cell–Derived Exosomes Recover Impaired Matrix Metabolism of Torn Human Rotator Cuff Tendons by Maintaining Tissue Homeostasis

Background: Adipose stem cell–derived exosomes (ASC-Exos) are reported to effectively prevent muscle atrophy and degeneration of torn rat rotator cuff, but their influence on human samples and their potential mechanism are still unclear. Purpose: We aimed to investigate the effects of ASC-Exos on the metabolic activities of torn human rotator cuff tendons and explore the potential mechanism behind it. Study Design: Controlled laboratory study. Methods: Diseased supraspinatus tendons were harvested from 15 patients with a mean ± SD age of 65.8 ± 3.2 years who underwent reverse shoulder arthroplasty for chronic rotator cuff tears associated with glenohumeral pathological changes. Each tendon was dissected into 3 × 4 × 4–mm explants: the ones derived from the same tendon were placed into 12-well plates and cultured in complete culture media (control) or in complete culture media supplemented with ASC-Exos for 72 hours. Afterward, the concentrations of cytokines secreted into the culture media—including interleukin 1β (IL-1β), IL-6, IL-8, and matrix metalloproteinase 9 (MMP-9)—were measured using enzyme-linked immunosorbent assay (ELISA). Tendons were stained with hematoxylin and eosin and immunohistochemistry (type I and III collagens) for histological analyses. Moreover, the expression of anabolic genes ( TIMP-1 and TIMP-3; type I and III collagen encoding) and catabolic genes ( MMP-9 and MMP-13) in tendons were measured using real-time quantitative polymerase chain reaction. Phosphorylated AMPKα and Wnt/β-catenin pathways were assayed by western blotting to explore the potential mechanism of action of ASC-Exos. Results: Secretion of proinflammatory cytokines, including IL-1β, IL-6, and MMP-9, was significantly reduced in the ASC-Exos group as compared with the control group. Supraspinatus tendons in the ASC-Exos group exhibited superior histological properties, as demonstrated by higher tendon maturing scores and more type I collagen content, but there was no significant difference in type III collagen content between groups. Expression of MMP-9 and MMP-13 genes was decreased in the ASC-Exos group versus the control group. Increased expression of type I and III collagens and an elevated type I/III ratio were found in the ASC-Exos group when compared with the control group. There was no significant difference in the secretion of IL-8 and expression of TIMP-1 and TIMP-3 genes between the ASC-Exos and control groups. Western blotting revealed that ASC-Exos enhanced phosphorylated AMPKα and decreased β-catenin levels to prevent tendon degeneration. Conclusion: ASC-Exos maintained metabolic homeostasis of torn human rotator cuff tendons to improve their histological properties, which might be achieved by enhancing AMPK signaling to suppress Wnt/β-catenin activity. Clinical Relevance: ASC-Exos could be used as an effective biological tool to promote healing in torn human rotator cuff tendons.

In vitro effects of alendronate on fibroblasts of the human rotator cuff tendon

Background Bone mineral density of the humeral head is an independent determining factor for postoperative rotator cuff tendon healing. Bisphosphonates, which are commonly used to treat osteoporosis, have raised concerns regarding their relationships to osteonecrosis of the jaw and to atypical fracture of the femur. In view of the prevalence of rotator cuff tear in osteoporotic elderly people, it is important to determine whether bisphosphonates affect rotator cuff tendon healing. However, no studies have investigated bisphosphonates’ cytotoxicity to human rotator cuff tendon fibroblasts (HRFs) or bisphosphonates’ effects on rotator cuff tendon healing. The purpose of this study was to evaluate the cytotoxicity of alendronate (Ald), a bisphosphonate, and its effects on HRF wound healing. Methods HRFs were obtained from human supraspinatus tendons, using primary cell cultures. The experimental groups were control, 0.1 μM Ald, 1 μM Ald, 10 μM Ald, and 100 μM Ald. Alendronate exposure was for 48 h, except during a cell viability analysis with durations from 1 day to 6 days. The experimental groups were evaluated for cell viability, cell cycle and cell proliferation, type of cell death, caspase activity, and wound-healing ability. Results The following findings regarding the 100 μM Ald group contrasted with those for all the other experimental groups: a significantly lower rate of live cells (p < 0.01), a higher rate of subG1 population, a lower rate of Ki-67 positive cells, higher rates of apoptosis and necrosis, a higher number of cells with DNA fragmentation, higher caspase-3/7 activity (p < 0.001), and a higher number of caspase-3 positive staining cells. In scratch-wound healing analyses of all the experimental groups, all the wounds healed within 48 h, except in the 100 μM Ald group (p < 0.001). Conclusions Low concentrations of alendronate appear to have little effect on HRF viability, proliferation, migration, and wound healing. However, high concentrations are significantly cytotoxic, impairing cellular proliferation, cellular migration, and wound healing in vitro.

Comparative Analysis of Platelet-rich Plasma Effect on Tenocytes from Normal Human Rotator Cuff Tendon and Human Rotator Cuff Tendon with Degenerative Tears

BACKGROUND: Platelet-rich plasma (PRP) stimulates cell proliferation and enhances matrix gene expression and synthesis. However, there have been no comparative study of the PRP effect on the normal and degenerative tenocytes. The purpose of this study was to compare the effect of PRP on tenocytes from normal and degenerative tendon.METHODS: Tendon tissues were obtained from patients undergoing arthroscopic repair (n=9) and from healthy donors (n=3). Tenocytes were cultured with 10% (vol/vol) platelet-poor plasma, PRP activated with calcium, and PRP activated with calcium and thrombin. The total cell number was assessed at days 7 and 14. The expressions of type I and III collagen, decorin, tenascin-C, and scleraxis were evaluated by quantitative real-time reverse transcriptase polymerase chain reaction. The total collagen and glycosaminoglycan (GAG) synthesis was evaluated at days 7 and 14.RESULTS: No differences were observed between the groups at day 7, but cell proliferation was remarkably increased in tenocytes from the degenerative tendon at day 14. In both tenocyte groups, the gene expressions of type I and III collagen were up-regulated. GAG synthesis was greater in the normal tendon, whereas the expressions of decorin and tenascin-C were increased in tenocytes from the degenerative tendon. Tenocytes from the degenerative tendon had higher fold-change of GAG synthesis and a lower collagen III/I ratio than normal tenocytes.CONCLUSIONS: PRP promoted the cell proliferation and enhanced the synthesis of tendon matrix in both groups. PRP has a greater positive effect on cell proliferation, matrix gene expression and synthesis in tenocytes from degenerative tendon.