Aging and the emerging role of cellular senescence in osteoarthritis.

OBJECTIVE: The correlation between age and incidence of osteoarthritis (OA) is well known but the causal mechanisms involved are not completely understood. This narrative review summarizes selected key findings from the past 30 years that have elucidated key aspects of the relationship between aging and OA. METHODS: The peer-reviewed English language literature was searched on PubMed using keywords including senescence, aging, cartilage, and osteoarthritis, for original studies and reviews published from 1993 to 2023 with a major focus on more recent studies. Manuscripts most relevant to aging and OA that examined one or more of the hallmarks of aging were selected for further review. RESULTS: All proposed hallmarks of aging have been observed in articular cartilage and some have also been described in other joint tissues. Hallmarks include genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, disabled macroautophagy, chronic inflammation, and dysbiosis. There is evidence that these age-related changes contribute to the development of OA in part by promoting cellular senescence. Senescence may therefore serve as a downstream mediator that connects numerous aging hallmarks to OA, likely through the senescence-associated secretory phenotype that is characterized by increased production of proinflammatory cytokines and matrix metalloproteinases. CONCLUSIONS: Progress over the past 30 years has provided the foundation for emerging therapies, such as senolytics and senomorphics, that hold promise for OA disease modification. Mechanistic studies utilizing physiologically-aged animals and cadaveric human joint tissues will be important for continued progress.

Use of a novel magnetically actuated compression system to study the temporal dynamics of axial and lateral strain in human osteochondral plugs.

The high water content of articular cartilage allows this biphasic tissue to withstand large compressive loads through fluid pressurization. The system presented here, termed the "MagnaSquish", provides new capabilities for quantifying the effect of rehydration on cartilage behavior during cyclic loading. An imbalanced rate of fluid exudation during load and fluid re-entry during recovery can lead to the accumulation of strain during successive loading cycles - a phenomenon known as ratcheting. Typical experimental systems for cartilage biomechanics use continuous contact between the platen and sample, which may affect tissue rehydration by compressing the top layer of cartilage and slowing fluid re-entry. To address this limitation, we developed a magnetically actuated device that provides full lift-off of the platen in between loading cycles. We investigated strain accumulation in cadaveric human osteochondral plugs during 750 loading cycles, with two dimensional profiles of the cartilage captured at 30 frames per second throughout loading and 10 min of additional free swelling recovery. Axial and lateral strain measurements were extracted from the tissue profiles using a UNet-based deep learning algorithm to circumvent manual tracing. We observed increased axial strain accumulation with shorter inter-cycle recovery, with static loading serving as the extreme case of zero recovery. The loading waveform during the 750 cycles dictated the pace of the recovery during the extended free swelling period, as shorter inter-cycle recovery led to more persistent axial strain accumulation for up to five minutes. This work showcases the importance of fluid re-entry in resisting strain accumulation during cyclical compression.

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

While advanced age is widely recognized as the greatest risk factor for osteoarthritis (OA), the biological mechanisms behind this connection remain unclear. Previous work has demonstrated that chondrocytes from older cadaveric donors have elevated levels of DNA damage as compared to chondrocytes from younger donors. The purpose of this study was to determine whether a decline in DNA repair efficiency is one explanation for the accumulation of DNA damage with age, and to quantify the improvement in repair with activation of Sirtuin 6 (SIRT6). After acute damage with irradiation, DNA repair was shown to be more efficient in chondrocytes from young (≤45 years old) as compared to middle-aged (50-65 years old) or older (>70 years old) cadaveric donors. Activation of SIRT6 with MDL-800 improved the repair efficiency, while inhibition with EX-527 reduced the rate of repair and increased the percentage of cells that retain high levels of damage. In addition to affecting repair after acute damage, treating chondrocytes from older donors with MDL-800 for 48 hours significantly reduced the amount of baseline DNA damage. Chondrocytes isolated from the knees of mice between 4 months and 22 months of age revealed both an increase in DNA damage with aging, and a decrease in DNA damage following MDL-800 treatment. Lastly, treating murine cartilage explants with MDL-800 lowered the percentage of chondrocytes with high p16 promoter activity, which supports the concept that using SIRT6 activation to maintain low levels of DNA damage may prevent the initiation of senescence.

Redox-active endosomes mediate α5ß1 integrin signaling and promote chondrocyte matrix metalloproteinase production in osteoarthritis.

Mechanical cues sensed by integrins induce cells to produce proteases to remodel the extracellular matrix. Excessive protease production occurs in many degenerative diseases, including osteoarthritis, in which articular cartilage degradation is associated with the genesis of matrix protein fragments that can activate integrins. We investigated the mechanisms by which integrin signals may promote protease production in response to matrix changes in osteoarthritis. Using a fragment of the matrix protein fibronectin (FN) to activate the α5ß1 integrin in primary human chondrocytes, we found that endocytosis of the integrin and FN fragment complex drove the production of the matrix metalloproteinase MMP-13. Activation of α5ß1 by the FN fragment, but not by intact FN, was accompanied by reactive oxygen species (ROS) production initially at the cell surface, then in early endosomes. These ROS-producing endosomes (called redoxosomes) contained the integrin-FN fragment complex, the ROS-producing enzyme NADPH oxidase 2 (NOX2), and SRC, a redox-regulated kinase that promotes MMP-13 production. In contrast, intact FN was endocytosed and trafficked to recycling endosomes without inducing ROS production. Articular cartilage from patients with osteoarthritis showed increased amounts of SRC and the NOX2 complex component p67phox. Furthermore, we observed enhanced localization of SRC and p67phox at early endosomes, suggesting that redoxosomes could transmit and sustain integrin signaling in response to matrix damage. This signaling mechanism not only amplifies the production of matrix-degrading proteases but also establishes a self-perpetuating cycle that contributes to the ongoing degradation of cartilage matrix in osteoarthritis.

Patient-derived extracellular matrix demonstrates role of COL3A1 in blood vessel mechanics.

Vascular Ehlers-Danlos Syndrome (vEDS) is a rare autosomal dominant disease caused by mutations in the COL3A1 gene, which renders patients susceptible to aneurysm and arterial dissection and rupture. To determine the role of COL3A1 variants in the biochemical and biophysical properties of human arterial ECM, we developed a method for synthesizing ECM directly from vEDS donor fibroblasts. We found that the protein content of the ECM generated from vEDS donor fibroblasts differed significantly from ECM from healthy donors, including upregulation of collagen subtypes and other proteins related to ECM structural integrity. We further found that ECM generated from a donor with a glycine substitution mutation was characterized by increased glycosaminoglycan content and unique viscoelastic mechanical properties, including increased time constant for stress relaxation, resulting in a decrease in migratory speed of human aortic endothelial cells when seeded on the ECM. Collectively, these results demonstrate that vEDS patient-derived fibroblasts harboring COL3A1 mutations synthesize ECM that differs in composition, structure, and mechanical properties from healthy donors. These results further suggest that ECM mechanical properties could serve as a prognostic indicator for patients with vEDS, and the insights provided by the approach demonstrate the broader utility of cell-derived ECM in disease modeling. STATEMENT OF SIGNIFICANCE: The role of collagen III ECM mechanics remains unclear, despite reported roles in diseases including fibrosis and cancer. Here, we generate fibrous, collagen-rich ECM from primary donor cells from patients with vascular Ehlers-Danlos syndrome (vEDS), a disease caused by mutations in the gene that encodes collagen III. We observe that ECM grown from vEDS patients is characterized by unique mechanical signatures, including altered viscoelastic properties. By quantifying the structural, biochemical, and mechanical properties of patient-derived ECM, we identify potential drug targets for vEDS, while defining a role for collagen III in ECM mechanics more broadly. Furthermore, the structure/function relationships of collagen III in ECM assembly and mechanics will inform the design of substrates for tissue engineering and regenerative medicine.

Epigenetics as a mediator of genetic risk in osteoarthritis: role during development, homeostasis, aging, and disease progression.

The identification of genomic loci that are associated with osteoarthritis (OA) has provided a starting point for understanding how genetic variation activates catabolic processes in the joint. However, genetic variants can only alter gene expression and cellular function when the epigenetic environment is permissive to these effects. In this review, we provide examples of how epigenetic shifts at distinct life stages can alter the risk for OA, which we posit is critical for the proper interpretation of genome-wide association studies (GWAS). During development, intensive work on the growth and differentiation factor 5 (GDF5) locus has revealed the importance of tissue-specific enhancer activity in controlling both joint development and the subsequent risk for OA. During homeostasis in adults, underlying genetic risk factors may help establish beneficial or catabolic "set points" that dictate tissue function, with a strong cumulative effect on OA risk. During aging, methylation changes and the reorganization of chromatin can "unmask" the effects of genetic variants. The destructive function of variants that alter aging would only mediate effects after reproductive competence and thus avoid any evolutionary selection pressure, as consistent with larger frameworks of biological aging and its relationship to disease. A similar "unmasking" may occur during OA progression, which is supported by the finding of distinct expression quantitative trait loci (eQTLs) in chondrocytes depending on the degree of tissue degradation. Finally, we propose that massively parallel reporter assays (MPRAs) will be a valuable tool to test the function of putative OA GWAS variants in chondrocytes from different life stages.

SIRT6 activation rescues the age-related decline in DNA damage repair in primary human chondrocytes.

While advanced age has long been recognized as the greatest risk factor for osteoarthritis (OA), the biological mechanisms behind this connection remain unclear. Previous work has demonstrated that chondrocytes from older cadaveric donors have elevated levels of DNA damage as compared to chondrocytes from younger donors. The purpose of this study was to determine whether a decline in DNA repair efficiency is one explanation for the accumulation of DNA damage with age, and to quantify the improvement in repair with activation of Sirtuin 6 (SIRT6). Using an acute irradiation model to bring the baseline level of all donors to the same starting point, this study demonstrates a decline in repair efficiency during aging when comparing chondrocytes from young (≤45 years old), middle-aged (50-65 years old), or older (>70 years old) cadaveric donors with no known history of OA or macroscopic cartilage degradation at isolation. Activation of SIRT6 in middle-aged chondrocytes with MDL-800 (20 µM) improved the repair efficiency, while inhibition with EX-527 (10 µM) inhibited the rate of repair and the increased the percentage of cells that retained high levels of damage. Treating chondrocytes from older donors with MDL-800 for 48 hours significantly reduced the amount of DNA damage, despite this damage having accumulated over decades. Lastly, chondrocytes isolated from the proximal femurs of mice between 4 months and 22 months of age revealed both an increase in DNA damage with aging, and a decrease in DNA damage following MDL-800 treatment.

3D chromatin structure in chondrocytes identifies putative osteoarthritis risk genes.

Genome-wide association studies have identified over 100 loci associated with osteoarthritis risk, but the majority of osteoarthritis risk variants are noncoding, making it difficult to identify the impacted genes for further study and therapeutic development. To address this need, we used a multiomic approach and genome editing to identify and functionally characterize potential osteoarthritis risk genes. Computational analysis of genome-wide association studies and ChIP-seq data revealed that chondrocyte regulatory loci are enriched for osteoarthritis risk variants. We constructed a chondrocyte-specific regulatory network by mapping 3D chromatin structure and active enhancers in human chondrocytes. We then intersected these data with our previously collected RNA-seq dataset of chondrocytes responding to fibronectin fragment, a known osteoarthritis trigger. Integration of the 3 genomic datasets with recently reported osteoarthritis genome-wide association study variants revealed a refined set of putative causal osteoarthritis variants and their potential target genes. One of the putative target genes identified was SOCS2, which was connected to a putative causal variant by a 170-kb loop and is differentially regulated in response to fibronectin fragment. CRISPR-Cas9-mediated deletion of SOCS2 in primary human chondrocytes from 3 independent donors led to heightened expression of inflammatory markers after fibronectin fragment treatment. These data suggest that SOCS2 plays a role in resolving inflammation in response to cartilage matrix damage and provides a possible mechanistic explanation for its influence on osteoarthritis risk. In total, we identified 56 unique putative osteoarthritis risk genes for further research and potential therapeutic development.

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

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.

Long-term treatment with senolytic drugs Dasatinib and Quercetin ameliorates age-dependent intervertebral disc degeneration in mice.

Intervertebral disc degeneration is highly prevalent within the elderly population and is a leading cause of chronic back pain and disability. Due to the link between disc degeneration and senescence, we explored the ability of the Dasatinib and Quercetin drug combination (D + Q) to prevent an age-dependent progression of disc degeneration in mice. We treated C57BL/6 mice beginning at 6, 14, and 18 months of age, and analyzed them at 23 months of age. Interestingly, 6- and 14-month D + Q cohorts show lower incidences of degeneration, and the treatment results in a significant decrease in senescence markers p16INK4a, p19ARF, and SASP molecules IL-6 and MMP13. Treatment also preserves cell viability, phenotype, and matrix content. Although transcriptomic analysis shows disc compartment-specific effects of the treatment, cell death and cytokine response pathways are commonly modulated across tissue types. Results suggest that senolytics may provide an attractive strategy to mitigating age-dependent disc degeneration.

Sirtuin 6 (SIRT6) regulates redox homeostasis and signaling events in human articular chondrocytes.

The nuclear localized protein deacetylase, SIRT6, has been identified as a crucial regulator of biological processes that drive aging. Among these processes, SIRT6 can promote resistance to oxidative stress conditions, but the precise mechanisms remain unclear. The objectives of this study were to examine the regulation of SIRT6 activity by age and oxidative stress and define the role of SIRT6 in maintaining redox homeostasis in articular chondrocytes. Although SIRT6 levels did not change with age, SIRT6 activity was significantly reduced in chondrocytes isolated from older adults. Using dimedone-based chemical probes that detect oxidized cysteines, we identified that SIRT6 is oxidized in response to oxidative stress conditions, an effect that was associated with reduced SIRT6 activity. Enhancement of SIRT6 activity through adenoviral SIRT6 overexpression specifically increased the basal levels of two antioxidant proteins, peroxiredoxin 1 (Prx1) and sulfiredoxin (Srx) and decreased the levels of an inhibitor of antioxidant activity, thioredoxin interacting protein (TXNIP). Conversely, in chondrocytes derived from mice with cartilage specific Sirt6 knockout, Sirt6 loss decreased Prx1 levels and increased TXNIP levels. SIRT6 overexpression decreased nuclear-generated H2O2 levels and oxidative stress-induced accumulation of nuclear phosphorylated p65. Our data demonstrate that SIRT6 activity is altered with age and oxidative stress conditions associated with aging. SIRT6 contributes to chondrocyte redox homeostasis by regulating specific members of the Prx catalytic cycle. Targeted therapies aimed at preventing the age-related decline in SIRT6 activity may represent a novel strategy to maintain redox balance in joint tissues and decrease catabolic signaling events implicated in osteoarthritis (OA).

Mechanisms and therapeutic implications of cellular senescence in osteoarthritis.

The development of osteoarthritis (OA) correlates with a rise in the number of senescent cells in joint tissues, and the senescence-associated secretory phenotype (SASP) has been implicated in cartilage degradation and OA. Age-related mitochondrial dysfunction and associated oxidative stress might induce senescence in joint tissue cells. However, senescence is not the only driver of OA, and the mechanisms by which senescent cells contribute to disease progression are not fully understood. Furthermore, it remains uncertain which joint cells and SASP-factors contribute to the OA phenotype. Research in the field has looked at developing therapeutics (namely senolytics and senomorphics) that eliminate or alter senescent cells to stop disease progression and pathogenesis. A better understanding of how senescence contributes to joint dysfunction may enhance the effectiveness of these approaches and provide relief for patients with OA.

Deletion of JNK Enhances Senescence in Joint Tissues and Increases the Severity of Age-Related Osteoarthritis in Mice.

OBJECTIVE: To determine the role of JNK signaling in the development of osteoarthritis (OA) induced by joint injury or aging in mice. METHODS: In the joint injury model, 12-week-old wild-type control, JNK1-/- , JNK2-/- , and JNK1fl/fl JNK2-/- aggecan-CreERT 2 double-knockout mice were subjected to destabilization of the medial meniscus (DMM) (n = 15 mice per group) or sham surgery (n = 9-10 mice per group), and OA was evaluated 8 weeks later. In the aging experiment, wild-type control, JNK1-/- , and JNK2-/- mice (n = 15 per group) were evaluated at 18 months of age. Mouse knee joints were evaluated by scoring articular cartilage structure, toluidine blue staining, osteophytes, and synovial hyperplasia, by histomorphometric analysis, and by immunostaining for the senescence marker p16INK 4a . Production of matrix metalloproteinase 13 (MMP-13) in cartilage explants in response to fibronectin fragments was measured by enzyme-linked immunosorbent assay. RESULTS: There were no differences after DMM surgery between the wild-type and the JNK-knockout mouse groups in articular cartilage structure, toluidine blue, or osteophyte scores or in MMP-13 production in explants. All 3 knockout mouse groups had increased subchondral bone thickness and area of cartilage necrosis compared to wild-type mice. Aged JNK-knockout mice had significantly worse articular cartilage structure scores compared to the aged wild-type control mice (mean ± SD 52 ± 24 in JNK1-/- mice and 60 ± 25 in JNK2-/- mice versus 32 ± 18 in controls; P = 0.02 and P = 0.004, respectively). JNK1-/- mice also had higher osteophyte scores. Deletion of JNK resulted in increased expression of p16INK 4a in the synovium and cartilage in older mice. CONCLUSION: JNK1 and JNK2 are not required for the development of OA in the mouse DMM model. Deletion of JNK1 or JNK2 is associated with more severe age-related OA and increased cell senescence, suggesting that JNK may act as a negative regulator of senescence in the joint.

Arp2/3 inactivation causes intervertebral disc and cartilage degeneration with dysregulated TonEBP-mediated osmoadaptation.

Extracellular matrix and osmolarity influence the development and homeostasis of skeletal tissues through Rho GTPase-mediated alteration of the actin cytoskeleton. This study investigated whether the actin-branching Arp2/3 complex, a downstream effector of the Rho GTPases Cdc42 and Rac1, plays a critical role in maintaining the health of matrix-rich and osmotically loaded intervertebral discs and cartilage. Mice with constitutive intervertebral disc- and cartilage-specific deletion of the critical Arp2/3 subunit Arpc2 (Col2-Cre; Arpc2fl/fl) developed chondrodysplasia and spinal defects. Since these mice did not survive to adulthood, we generated mice with inducible Arpc2 deletion in disc and cartilage (Acan-CreERT2; Arpc2fl/fl). Inactivation of Arp2/3 at skeletal maturity resulted in growth plate closure, loss of proteoglycan content in articular cartilage, and degenerative changes in the intervertebral disc at 1 year of age. Chondrocytes with Arpc2 deletion showed compromised cell spreading on both collagen and fibronectin. Pharmacological inhibition of Cdc42 and Arp2/3 prevented the osmoadaptive transcription factor TonEBP/NFAT5 from recruiting cofactors in response to a hyperosmolarity challenge. Together, these findings suggest that Arp2/3 plays a critical role in cartilaginous tissues through the regulation of cell-extracellular matrix interactions and modulation of TonEBP-mediated osmoadaptation.

Microfluidics for the study of mechanotransduction.

Mechanical forces regulate a diverse set of biological processes at cellular, tissue, and organismal length scales. Investigating the cellular and molecular mechanisms that underlie the conversion of mechanical forces to biological responses is challenged by limitations of traditional animal models and in vitro cell culture, including poor control over applied force and highly artificial cell culture environments. Recent advances in fabrication methods and material processing have enabled the development of microfluidic platforms that provide precise control over the mechanical microenvironment of cultured cells. These devices and systems have proven to be powerful for uncovering and defining mechanisms of mechanotransduction. In this review, we first give an overview of the main mechanotransduction pathways that function at sites of cell adhesion, many of which have been investigated with microfluidics. We then discuss how distinct microfluidic fabrication methods can be harnessed to gain biological insight, with description of both monolithic and replica molding approaches. Finally, we present examples of how microfluidics can be used to apply both solid forces (substrate mechanics, strain, and compression) and fluid forces (luminal, interstitial) to cells. Throughout the review, we emphasize the advantages and disadvantages of different fabrication methods and applications of force in order to provide perspective to investigators looking to apply forces to cells in their own research.

Engineered Cartilage from Human Chondrocytes with Homozygous Knockout of Cell Cycle Inhibitor p21.

Osteoarthritis (OA) is a highly prevalent disease with limited treatment options. The search for disease-modifying OA therapies would benefit from a more comprehensive knowledge of the genetic variants that contribute to chondrocyte dysfunction and the barriers to cartilage regeneration. One goal of this study was to establish a system for producing engineered cartilage tissue from genetically defined primary human chondrocytes through genome editing and single-cell expansion. This process was utilized to investigate the functional effect of biallelic knockout of the cell cycle inhibitor p21. The use of ribonucleoprotein (RNP) CRISPR/Cas9 complexes targeting two sites in the coding region of p21 resulted in a high frequency (16%) of colonies with homozygous p21 knockout. Chondrogenic pellet cultures from expanded chondrocytes with complete loss of p21 produced more glycosaminoglycans (GAG) and maintained a higher cell number. Single-cell-derived colonies retained the potential for robust matrix production after expansion, allowing for analysis of colony variability from the same population of targeted cells. The effect of enhanced cartilage matrix production in p21 knockout chondrocytes persisted when matrix production from individual colonies was analyzed. Chondrocytes had lower levels of p21 protein with further expansion, and the difference in GAG production with p21 knockout was strongest at early passages. These results support previous findings that implicate p21 as a barrier to cartilage matrix production and regenerative capacity. Furthermore, this work establishes the use of genome-edited human chondrocytes as a promising approach for engineered tissue models containing user-defined gene knockouts and other genetic variants for investigation of OA pathogenesis. Impact Statement This work provides two important advances to the field of tissue engineering. One is the demonstration that engineered cartilage tissue can be produced from genetically defined populations of primary human chondrocytes. While CRISPR/Cas-9 genome editing has been extensively used in cell lines that divide indefinitely, this work extends the technique to an engineered tissue model system to support investigation of genetic changes that affect cartilage production. A second contribution is the finding that chondrocytes with p21 knockout synthesized more cartilage matrix tissue than unedited controls. This supports the continued investigation of p21 as a potential barrier to effective cartilage regeneration.

Controlled induction and targeted elimination of p16INK4a-expressing chondrocytes in cartilage explant culture.

Cellular senescence is a phenotypic state that contributes to age-related diseases through the secretion of matrix-degrading and inflammatory molecules. An emerging therapeutic strategy for osteoarthritis (OA) is to selectively eliminate senescent cells by initiating apoptosis. This study establishes a cartilage explant model of senescence induction and senolytic clearance using p16Ink4a expression as a biomarker of senescence. Growth-factor stimulation of explants increased the expression of p16Ink4a at both the mRNA and protein levels. Applying this culture system to cartilage from p16tdTom reporter mice (a knockin allele with tdTomato fluorescent protein regulated by the endogenous p16Ink4a promoter) demonstrated the emergence of a p16-high population that was quantified using flow cytometry for tdTomato. Cell sorting was used to separate chondrocytes based on tdTomato fluorescence and p16-high cells showed higher senescence-associated ß-galactosidase activity and increased gene expression of the senescence-associated secretory phenotype as compared with p16-low cells. The potential for effective senolysis within the cartilage extracellular matrix was assessed using navitoclax (ABT-263). Navitoclax treatment reduced the percentage of p16-high cells from 17.9 to 6.1% (mean of 13 matched pairs; P < 0.001) and increased cleaved caspase-3 confirmed apoptotic activity. Together, these findings establish a physiologically relevant cartilage explant model for testing the induction and elimination of senescent chondrocytes, which will support investigations of senolytic therapy for OA.-Sessions, G. A., Copp, M. E., Liu, J.-Y., Sinkler, M. A., D'Costa, S., Diekman, B. O. Controlled induction and targeted elimination of p16INK4a-expressing chondrocytes in cartilage explant culture.