@article{copp_chubinskaya_bracey_shine_sessions_loeser_diekman_2022, title={Comet assay for quantification of the increased DNA damage burden in primary human chondrocytes with aging and osteoarthritis}, ISSN={["1474-9726"]}, DOI={10.1111/acel.13698}, abstractNote={Abstract}, journal={AGING CELL}, author={Copp, Michaela E. and Chubinskaya, Susan and Bracey, Daniel N. and Shine, Jacqueline and Sessions, Garrett and Loeser, Richard F. and Diekman, Brian O.}, year={2022}, month={Aug} }
 @article{copp_flanders_gagliardi_gilbertie_sessions_chubinskaya_loeser_schnabel_diekman_2021, title={The combination of mitogenic stimulation and DNA damage induces chondrocyte senescence}, volume={29}, ISSN={["1522-9653"]}, DOI={10.1016/j.joca.2020.11.004}, abstractNote={Cellular senescence is a phenotypic state characterized by stable cell-cycle arrest, enhanced lysosomal activity, and the secretion of inflammatory molecules and matrix degrading enzymes. Senescence has been implicated in osteoarthritis (OA) pathophysiology; however, the mechanisms that drive senescence induction in cartilage and other joint tissues are unknown. While numerous physiological signals are capable of initiating senescence, one emerging theme is that damaged cells convert to senescence in response to sustained mitogenic stimulation. The goal of this study was to develop an in vitro articular cartilage explant model to investigate the mechanisms of senescence induction.This study utilized healthy cartilage derived from cadaveric equine stifles and human ankles. Explants were irradiated to initiate DNA damage, and mitogenic stimulation was provided through serum-containing medium and treatment with transforming growth factor β1 and basic fibroblastic growth factor. Readouts of senescence were a quantitative flow cytometry assay to detect senescence-associated β galactosidase activity (SA-β-gal), immunofluorescence for p16 and γH2AX, and qPCR for the expression of inflammatory genes.Human cartilage explants required both irradiation and mitogenic stimulation to induce senescence as compared to baseline control conditions (7.16% vs 2.34% SA-β-gal high, p = 0.0007). These conditions also resulted in chondrocyte clusters within explants, a persistent DNA damage response, increased p16, and gene expression changes.Treatment of cartilage explants with mitogenic stimuli in the context of cellular damage reliably induces high levels of SA-β-gal activity and other senescence markers, which provides a physiologically relevant model system to investigate the mechanisms of senescence induction.}, number={3}, journal={OSTEOARTHRITIS AND CARTILAGE}, author={Copp, M. E. and Flanders, M. C. and Gagliardi, R. and Gilbertie, J. M. and Sessions, G. A. and Chubinskaya, S. and Loeser, R. F. and Schnabel, L. and Diekman, B. O.}, year={2021}, month={Mar}, pages={402–412} }
 @article{sessions_sinkler_copp_diekman_2019, title={CONTROLLED INDUCTION AND TARGETED ELIMINATION OF P16(INK4A)-HIGH CHONDROCYTES TO INVESTIGATE SENESCENCE-MEDIATED CARTILAGE DYSFUNCTION}, volume={27}, ISSN={["1522-9653"]}, DOI={10.1016/j.joca.2019.02.140}, abstractNote={Purpose: Cellular senescence is a phenotypic state that emerges in response to stress after injury and during aging. Senescent cells are characterized by increased expression of the cell cycle inhibitor p16INK4a (p16), high lysosomal activity, and the production of pro-inflammatory and matrix-degrading proteins known as the senescence associated secretory phenotype (SASP). Expression of p16 increases in both murine and human chondrocytes with age and high p16 expression correlates with SASP factor production. Furthermore, treatment with “senolytic” compounds that specifically target senescent cells for apoptosis was shown to mitigate post-traumatic OA in a murine model. The aim of this study was to establish a cartilage explant model system to explore the functional effects of selectively eliminating p16-expressing (senescent) chondrocytes. Methods: A knock-in murine genetics approach was used to replace the coding region of p16 with the fluorescent molecule tdTomato in order to quantify p16 expression on a single-cell basis with flow cytometry. Femoral cap cartilage explants were isolated from mice with one intact copy of p16 and one copy of the p16tdTomato allele. Explants were cultured for three weeks either in control media or senescence induction media containing 1 ng/ml transforming growth factor beta-1 and 5 ng/ml basic fibroblastic growth factor. Explants were digested and the percentage of p16tdTomato-high cells was determined by flow cytometry. Cell sorting was used to separate chondrocytes into p16tdTomato-high and p16tdTomato-low fractions and these populations were analyzed for senescence features such as senescence-associated beta galactosidase (SABG) activity. Senescent cells upregulate anti-apoptotic pathways to maintain survival despite significant cellular damage. The ability to selectively eliminate p16-high cells with a drug that inhibits this apoptotic block is both a potential treatment and a way to further confirm the senescent nature of the p16tdTomato-high cell fraction. In this study, senolytic treatment was performed using navitoclax, a BCL pathway inhibitor, at a concentration of 5 μM for 72 hours. As an initial assessment of how senolytic clearance affects cartilage health, the glycosaminoglycans (GAGs) content was measured in femoral cap explants that were cultured for an additional week following control or navitoclax treatment. Results: The treatment of murine cartilage explants with growth factors for three weeks induces a significant fraction of p16tdTomato-high chondrocytes (Figure 1). This p16tdTomato-high cell population demonstrates higher SABG activity as compared to the p16tdTomato-low fraction (n=4 sorts, mean = 39.1% vs. 20.5% positive, p=0.03 by paired t-test). Following treatment with the senolytic navitoclax, explants showed a significant reduction in the percentage of p16tdTomato-high cells (Figure 2). As compared to control treatment, clearing senescent cells with navitoclax shows a trend towards increasing the GAG content per cell in cartilage explants (n=4 matched explants, mean = 57.6 vs. 41.3 μg GAG/μg DNA, p=0.06 by paired t-test). Conclusions: This work utilized a p16-based reporter allele to quantify the induction and elimination of senescent chondrocytes. As consistent with known conditions for senescence induction, the percentage of p16tdTomato-high cells increased upon growth factor stimulation in the context of growth restraint provided by the extra-cellular matrix. These p16tdTomato-high cells showed features of senescence and were selectively eliminated upon treatment with navitoclax. Analysis in a set of matched cartilage explants indicates that senolytic treatment is capable of increasing GAG levels, which suggests that senescence clearance may improve matrix homeostasis and limit cartilage degradation associated with aging and joint injury.View Large Image Figure ViewerDownload Hi-res image Download (PPT)}, journal={OSTEOARTHRITIS AND CARTILAGE}, author={Sessions, G. A. and Sinkler, M. A. and Copp, M. E. and Diekman, B. O.}, year={2019}, month={Apr}, pages={S97–S97} }