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    Orthopedic and Rehabilitation Engineering

    (G-265) Comparing Senescence Induction Methods In Primary Murine Tenocytes

    Thursday, October 12, 2023
    2:00 PM – 3:00 PM PDT
    Location: Exhibit Hall - Row G - Poster # 265
    Introduction:: Musculoskeletal disorders, including tendinopathy, are a prevalent problem in the aging population. Aging tissue is associated with an increasing number of senescent cells [1]. Features of cellular senescence vary by cell type and induction method; common features include cell cycle arrest, DNA damage (γH2AX), senescence-associated beta-galactosidase (SA-β-Gal) expression, and a pro-inflammatory senescence-associated secretory phenotype (SASP) [2,3]. Given the importance of studying cellular senescence, researchers have examined multiple methods of inducing senescence in vitro in various cell types, but not in primary murine tenocytes to date [3]. Tenocytes are primary tendon cells [1]. We sought to evaluate the effectiveness of inducing senescence in primary murine tenocytes, using two induction methods: irradiation and doxorubicin. We hypothesized that both methods would effectively induce senescence in tenocytes.

    Materials and Methods::  Flexor digitorum longus (FDL) tendons were isolated from 4-month-old male C57BL/6J mice (Young). Tendons were enzymatically digested to isolate primary tenocytes and expanded to P2 [4]. Cells were cultured with standard culture medium (low-glucose DMEM + 10% fetal bovine serum + 1% pen strep) at 37°C with medium changes every two days. Senescence was induced by addition of 200nM doxorubicin (DOX) to cell culture media for 72 hours or by 10Gy irradiation exposure (RAD). After 7 days, we assessed proliferation via 3H- thymidine incorporation and metabolic activity via resazurin reduction [5]. Cells were plated onto poly-d-lysine coated coverslips and stained with SA-β-Gal, γH2AX, and DAPI. Images were captured on Olympus IX83 inverted microscope and custom software was used to quantify positively stained cells. Statistical analysis was completed using one-way ANOVas with significance set at *p< 0.05, trends at p< 0.1.

    Results, Conclusions, and Discussions:: There is a significant decrease in proliferation of DOX tenocytes and a trend of decreased proliferation in RAD tenocytes compared to young (Fig. 1F). DOX and RAD also had significantly more cells with DNA damage and more γH2AX foci per cell (Fig. 1A,C,E). Only DOX had significantly more cells with SA-β-Gal expression while RAD had a trending increase (Fig. 1A, B). No significant changes were found in metabolic activity (Fig. 1G).While both RAD and DOX induced senescence, DOX treatment produced higher numbers of senescent tenocytes. RAD groups had greater variability, which is likely due to other variables that interfere with irradiation, such as vessel size and shape [6]. Our work shows that 200nM DOX produced more senescent tenocytes than 10Gy irradiation. Future work focuses on characterizing the SASP factors and cell-specific biomarkers of senescent tenocytes and exploring mechanisms of secondary senescence. Ultimately, we aim to identify the effect of senescent cells on tendon physiology and their role in age-related tendon degeneration.

    Acknowledgements (Optional): : Research reported in this publication was supported by the Boston University Micro and Nano Imaging Facility and Undergraduate Research Opportunities Program.

    References (Optional): : [1] Tsai, W. J. Orthopaedic Research. 2011 Oct; 29:1598-1603. [2] Admasu, T. Ageing Res.Rev. 2021 Vol. 70, 10142. [3] Hernandez-Segura, A. Trends Cell Biol. 2018 Vol. 28, 6:436-53 [4] Paredes, J. FASEB J. 2020 Jun; 34(6): 8341-56 [5] Connizzo, B. Connect. Tissue Res. 2020 Vol. 61,1:48-62. [6] Benadjaoud, M. Int J. Radiat. Oncol. 2021 Vol. 112, 4:975-85