Tier 3 — preclinical

Sirtuin 6 activation rescues the age-related decline in DNA damage repair in primary human chondrocytes

Copp ME, Shine J, Brown HL, Nimmala KR, Hansen OB, Chubinskaya S, Collins JA, Loeser RF, Diekman BO

Aging (Albany NY) 2023 Volume 15, Issue 23, Pages 13628–13645

Bibliography

DOI: 10.18632/aging.205394
PubMed: PMID 38078876
PubMed Central: PMC10756124
Funding: Support provided by National Institutes of Health: R56 AG066911 to B.O.D.; R01 AG044034 to R.F.L. Procurement of human tissue supported in part by the Rush University Klaus Kuettner Endowed Chair for Research on Osteoarthritis (S.C.). Human tissue supplied by the Gift of Hope Tissue and Organ Donor Bank.
Competing interests: None declared.

Study snapshot

Design	Interventional ex vivo study using primary human chondrocytes from cadaveric ankle cartilage across three age groups, subjected to acute irradiation-induced DNA damage and quantified by alkaline comet assay across a 4-hour repair time course, with SIRT6 modulation by MDL-800 (activator) or EX-527 (inhibitor). Parallel murine chondrocyte study across four ages with 48-hour SIRT6 activation.
Model	Primary human chondrocytes isolated from ankle cartilage of cadaveric donors without OA history (Collins grade 0–2); young ≤45 years, middle-aged 50–65 years, older >70 years. Primary murine chondrocytes isolated from the proximal femur cartilage of C57BL/6 mice aged 4, 8, 14, and 22 months (note: the published version describes the murine source as knees).
Sample	Human cohorts: n=3 young donors, n=4 middle-aged donors, n=3 older donors for the age-comparison arm; n=8 middle-aged donors for the SIRT6 modulator arm; n=4 older donors for the MDL-800 accumulated-damage reduction arm. Murine cohorts: n=3 mice per age group. Approximately 100 cells per condition analysed by comet assay.
Intervention	Irradiation-induced acute DNA damage (10 Gy X-ray); SIRT6 activation with MDL-800 (20 μM); SIRT6 inhibition with EX-527 (10 μM); DMSO vehicle control. Repair kinetics measured at 15, 30, 45, 60, 120, and 240 minutes. Accumulated-damage arm used 48-hour 20 μM MDL-800 treatment without irradiation.
Duration	Acute (4-hour repair time course post-irradiation with 2-hour pre-treatment); chronic (48-hour SIRT6 modulator treatment for accumulated damage arm).
Endpoints	DNA damage as percentage of DNA in comet tails (alkaline comet assay, single-cell); Time course of DNA damage repair over 4 hours post-irradiation; Percentage of cells with high DNA damage (>60% DNA in comet tails) at 4 hours; Percentage of cells with low DNA damage (<15% DNA in comet tails) at 4 hours; Effect of SIRT6 activation (MDL-800) on repair efficiency; Effect of SIRT6 inhibition (EX-527) on repair efficiency; Reduction of accumulated DNA damage in older human chondrocytes after 48-hour MDL-800; Age-related DNA damage accumulation in murine chondrocytes across 4–22 months; Reduction of DNA damage in aged murine chondrocytes after 48-hour MDL-800

What the study showed, in plain terms

Chondrocytes — the cells that maintain cartilage — accumulate DNA damage as we age, and older chondrocytes carry substantially more damage than younger ones. This paper asked whether that accumulation is caused by chondrocytes becoming less efficient at repairing damage, and whether pharmacologically activating SIRT6 could rescue the deficit.

The team used cartilage from cadaveric ankle donors across three age groups (≤45, 50–65, and >70 years) and applied a controlled dose of irradiation to bring every donor's chondrocytes to the same starting level of damage. They then measured how quickly the cells repaired the damage over four hours using the alkaline comet assay. Younger donors' chondrocytes repaired the damage nearly completely. Middle-aged and older donors' cells repaired more slowly, and a substantial fraction of older-donor cells failed to repair at all — retaining very high damage burden at 4 hours.

Activating SIRT6 with the small molecule MDL-800 improved repair efficiency in middle-aged chondrocytes. Inhibiting SIRT6 with EX-527 slowed repair and increased the proportion of cells that got stuck with high damage. In older donors' chondrocytes, 48 hours of MDL-800 treatment cut accumulated DNA damage roughly in half without any irradiation — the equivalent of erasing approximately three decades of damage accumulation. The same pattern held in aged mouse chondrocytes.

The practical implication is that DNA damage in ageing chondrocytes is not a static, permanent mark; it can be reduced by activating an enzyme the body already has. Whether this translates into a way to slow or prevent osteoarthritis is the next question.

Key findings

Immediately after 10 Gy irradiation, chondrocytes from young (≤45), middle-aged (50–65), and older (>70) donors had similar levels of DNA damage, confirming that the irradiation dose overrode baseline age-related differences and enabled fair comparison of repair kinetics.
Over 4 hours, younger-donor chondrocytes reduced their DNA-in-comet-tail levels close to baseline; middle-aged and older-donor cells did not. At 60, 120, and 240 minutes, older-donor cells retained significantly more damage than middle-aged and/or young cells.
At 4 hours post-irradiation, 27.6% of older-donor chondrocytes retained high damage (>60% DNA in comet tails), versus 12.5% of middle-aged and 2.6% of young cells — the age-related repair deficit is driven by a bifurcated subpopulation of cells that fail to repair, not a uniform slowing.
MDL-800 (20 μM SIRT6 activator) applied to middle-aged chondrocytes improved repair kinetics across the time course (repeated-measures two-way ANOVA main effect p = 0.005 vs DMSO).
EX-527 (10 μM SIRT6 inhibitor) significantly increased DNA damage at baseline and throughout the repair time course; 37.2% of EX-527-treated cells retained high damage at 4 hours, versus far fewer in DMSO or MDL-800 groups.
MDL-800 improved repair in older-donor chondrocytes at 30, 60, 120, and 240 minutes post-irradiation, and cut the percentage of cells with high damage at 4 hours from 20.1% to 4.9%.
In older-donor chondrocytes not subjected to irradiation, 48 hours of MDL-800 reduced mean DNA-in-comet-tail from 21.3% to 11.3% (paired t-test p = 0.0031) — clearing accumulated damage that had built up over decades.
Murine chondrocytes from the C57BL/6 proximal femur showed DNA damage roughly doubling from 4 to 22 months of age. MDL-800 significantly reduced damage at 8, 14, and 22 months.
The scale of MDL-800's effect on older-donor human chondrocytes corresponds to erasing roughly 34 years of age-related damage accumulation, based on a linear regression from prior work by the same group linking chondrocyte age to comet-tail damage.

What this study can and cannot tell us

All results are at the chondrocyte level in vitro (ex vivo from cadaveric donors or freshly isolated from mice). The paper does not measure downstream cartilage-tissue outcomes (matrix integrity, joint function, OA incidence) or organism-level effects. Improved DNA repair in isolated chondrocytes is a promising surrogate but not proof that MDL-800 or any SIRT6 activator prevents osteoarthritis.

Human donors were selected for absence of macroscopic OA (Collins grade 0–2). The paper does not address whether the same repair rescue would occur in chondrocytes from cartilage that already shows OA changes.

The irradiation model creates an acute high-dose DNA damage burden that is useful for kinetics comparisons but does not replicate the low-level chronic damage that accumulates during ageing. The 48-hour MDL-800 experiments in older-donor cells and in aged mice do address accumulated damage more directly and are the strongest translational signal in the paper.

The alkaline comet assay measures single-strand breaks, double-strand breaks, and abasic sites collectively; it does not distinguish which lesion type accumulates or is preferentially repaired.

MDL-800 is a research-grade SIRT6 allosteric activator, not a supplement or approved therapy. The paper does not report whether dietary or supplement-form SIRT6 activators (fucoidan, cyanidin, quercetin) would produce comparable effects.