Comet assay for quantification of the increased DNA damage burden in primary human chondrocytes with aging and osteoarthritis

Abstract

It is known that chondrocytes from joints with osteoarthritis (OA) exhibit high levels of DNA damage, but the degree to which chondrocytes accumulate DNA damage during “normal aging” has not been established. The goal of this study was to quantify the DNA damage present in chondrocytes obtained from cadaveric donors of a wide age range, and to compare the extent of this damage to OA chondrocytes. The alkaline comet assay was used to measure the DNA damage in normal cartilage from the ankle (talus) and the knee (femur) of cadaveric donors, as well as in OA chondrocytes obtained at the time of total knee replacement. Chondrocytes from younger donors (<45 years) had less DNA damage than older donors (>70 years) as assessed by the percentage of DNA in the comet “tail”. In donors between 50 and 60 years old, there was increased DNA damage in chondrocytes from OA cartilage as compared to cadaveric. Talar chondrocytes from 23 donors between the ages of 34 and 78 revealed a linear increase in DNA damage with age (R2 = 0.865, p < 0.0001). A “two‐tailed” comet assay was used to demonstrate that most of the accumulated damage is in the form of strand breaks as opposed to alkali‐labile base damage. Chondrocytes from young donors required 10 Gy irradiation to recapitulate the DNA damage present in chondrocytes from older donors. Given the potential for DNA damage to contribute to chondrocyte dysfunction and senescence, this study supports the investigation of mechanisms by which hypo‐replicative cell types accumulate high levels of damage.

Adverse Effect of Blue Light on DNA Integrity Is Accelerated by 5-Aminolevulinic Acid in HaCaT Human Keratinocyte Cells and B16F1 Murine Melanoma Cells

“…We also demonstrated that the TT-comet assay could detect DSB and SSB formation separately in non-sperm cells. Recently, Copp et al, assessed DNA strand breaks in chondrocytes from cadaveric doners using the TT-comet assay [ 27 ]. Therefore, this assay may be applicable for the detection of both types of strand breaks in other cell types and tissues.…”

Single-cell protein activity analysis reveals a novel subpopulation of chondrocytes and the corresponding key master regulator proteins associated with anti-senescence and OA progression

“…It is well known that the DNA repair signaling pathway is crucial for the maintenance of chondrocyte homeostasis and anti-senescence, especially during aging ( 51 ). Previous studies have shown that TSPYL2 plays an important role in DNA damage repair and revealed the molecular mechanisms whereby TSPYL2 regulates SIRT1 and p53 activity upon DNA damage ( 35 , 52 ).…”

Role of endocrine PACAP in age-related diseases

“…Age-related diseases of the musculoskeletal system are important determinants of quality of life in old age. Our current understanding is that cell autonomus and nonautonomus pathways interact to regulate aging processes in the bone, joints and skeletal muscle, which are also modulated by lifestyle factors (108)(109)(110)(111)(112). PACAP had a positive effect on chondrogenesis and bone formation in both in vivo and in vitro studies.…”