Oxidative damage is a well-established driver of aging. Evidence of oxidative stress exists in aged and degenerated discs, but it is unclear how it affects disc metabolism. In this study, we first determined whether oxidative stress negatively impacts disc matrix metabolism using disc organotypic and cell cultures. Mouse disc organotypic culture grown at atmospheric oxygen (20% O2) exhibited perturbed disc matrix homeostasis, including reduced proteoglycan synthesis and enhanced expression of matrix metalloproteinases, compared to discs grown at low oxygen levels (5% O2). Human disc cells grown at 20% O2 showed increased levels of mitochondrial-derived superoxide anions and perturbed matrix homeostasis. Treatment of disc cells with the mitochondria-targeted reactive oxygen species (ROS) scavenger XJB-5-131 blunted the adverse effects caused by 20% O2. Importantly, we demonstrated that treatment of accelerated aging Ercc1−/Δmice, previously established to be a useful in vivo model to study age-related intervertebral disc degeneration (IDD), also resulted in improved disc total glycosaminoglycan content and proteoglycan synthesis. This demonstrates that mitochondrial-derived ROS contributes to age-associated IDD in Ercc1−/Δmice. Collectively, these data provide strong experimental evidence that mitochondrial-derived ROS play a causal role in driving changes linked to aging-related IDD and a potentially important role for radical scavengers in preventing IDD.

The collagen architecture in normal, aging, and osteoarthritic articular cartilage was studied optically using a new silver staining technique based on specimens from 50 autopsy cases, four amputated limbs, and six osteoarthritic knees. In the normal articular cartilage, the collagen fibrils in the superficial zone were compactly arranged into layers of decussating flat ribbons mostly parallel to the artificial split lines. The fibrils showed a tendency to condense into vertical arcade columns undergirded by tangential bundles in the intermediate zone. In the deep zone, the fibrils formed a random meshwork with a slight preponderance of vertical fibrils in the perilacunar region. Three types of early degradative lesions involving the collagen network were identified. Type I lesions consisted of focal superficial disruptions related to age and friction. Type II lesions consisted of focal disruptions of tangential fibrils in the intermediate zone leading to cyst formation, probably representing a form of local stress failure. Type III lesions were found in the patella and consisted of marked swelling of the superficial zone, the cause of which was unknown. Lesions of varying severity were seen within each of the three types; the morphological changes of the more severe lesions overlapped with those of clinically overt osteoarthritis.

BACKGROUND CONTEXT Tobacco smoking is a key risk factor for spine degeneration. However, the underlying mechanism by which smoking induces degeneration is not known. Recent studies implicate DNA damage as a cause of spine and intervertebral disc degeneration. Because tobacco smoke contains many genotoxins, we hypothesized that tobacco smoking promotes spine degeneration by inducing cellular DNA damage. PURPOSE To determine if DNA damage plays a causal role in smoking-induced spine degeneration. STUDY DESIGN To compare the effect of chronic tobacco smoke inhalation on intervertebral disc and vertebral bone in normal and DNA repair-deficient mice to determine the contribution of DNA damage to degenerative changes. METHODS Two month-old wild-type (C57BL/6) and DNA repair-deficient Ercc1−/Δ mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 7 weeks) to model first-hand smoking in humans. Total disc proteoglycan (PG) content (1,9-dimethylmethylene blue assay), PG synthesis (35S-sulfate incorporation assay), aggrecan proteolysis (immunoblotting analysis) and vertebral bone morphology (micro-computed tomography) were measured. RESULTS Exposure of wild-type mice to tobacco smoke led to a 19% increase in vertebral porosity and a 61% decrease in trabecular bone volume. Intervertebral discs of smoke-exposed animals also showed a 2.6-fold decrease in GAG content and a 8.1-fold decrease in new PG synthesis. These smoking-induced degenerative changes were similar but not worse in Ercc1−/Δ mice. CONCLUSIONS Short-term exposure to high levels of primary tobacco smoke inhalation promotes degeneration of vertebral bone and discs. Disc degeneration is primarily driven by reduced synthesis of proteoglycans needed for vertebral cushioning. Degeneration was not exacerbated in congenic DNA repair-deficient mice indicating that DNA damage per se does not have a significant causal role in driving smoke-induced spine degeneration.

Aging greatly increases the risk for intervertebral disc degeneration (IDD) as a result of proteoglycan loss due to reduced synthesis and enhanced degradation of the disc matrix proteoglycan (PG). How disc matrix PG homeostasis becomes perturbed with age is not known. The goal of this study is to determine whether cellular senescence is a source of this perturbation. We demonstrated that disc cellular senescence is dramatically increased in the DNA repair-deficient Ercc1−/Δ mouse model of human progeria. In these accelerated aging mice, increased disc cellular senescence is closely associated with the rapid loss of disc PG. We also directly examine PG homeostasis in oxidative damage-induced senescent human cells using an in vitro cell culture model system. Senescence of human disc cells treated with hydrogen peroxide was confirmed by growth arrest, senescence-associated β-galactosidase activity, γH2AX foci, and acquisition of senescence-associated secretory phenotype. Senescent human disc cells also exhibited perturbed matrix PG homeostasis as evidenced by their decreased capacity to synthesize new matrix PG and enhanced degradation of aggrecan, a major matrix PG. of the disc. Our in vivo and in vitro findings altogether suggest that disc cellular senescence is an important driver of PG matrix homeostatic perturbation and PG loss.

Intervertebral disc degeneration (IDD) is the leading cause of debilitating spinal disorders such as chronic lower back pain. Aging is the greatest risk factor for IDD. Previously, we demonstrated IDD in a murine model of a progeroid syndrome caused by reduced expression of a key DNA repair enzyme. This led us to hypothesize that DNA damage promotes IDD. To test our hypothesis, we chronically exposed adult wild-type (Wt) and DNA repair-deficient Ercc1−/Δ mice to the cancer therapeutic agent mechlorethamine (MEC) or ionization radiation (IR) to induce DNA damage and measured the impact on disc structure. Proteoglycan, a major structural matrix constituent of the disc, was reduced 3-5x in the discs of MEC- and IR-exposed animals compared to untreated controls. Expression of the protease ADAMTS4 and aggrecan proteolytic fragments were significantly increased. Additionally, new PG synthesis was reduced 2-3x in MEC- and IR-treated discs compared to untreated controls. Both cellular senescence and apoptosis were increased in discs of treated animals. The effects were more severe in the DNA repair-deficient Ercc1−/Δ mice than in Wt littermates. Local irradiation of the vertebra in Wt mice elicited a similar reduction in PG. These data demonstrate that genotoxic stress drives degenerative changes associated with IDD.

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Tobacco smoking increases the risk of intervertebral disc degeneration (IDD) and back pain, but the mechanisms underlying the adverse effects of smoking are largely unknown. Current hypotheses predict that smoking contributes to IDD indirectly through nicotine-mediated vasoconstriction which limits the exchange of nutrients between the discs and their surroundings. We alternatively hypothesize that direct contact of disc cells, that is, cells in the outermost annulus and those present along fissures in degenerating discs, with the vascular system containing soluble tobacco smoking constituents could perturb normal metabolic activities resulting in IDD. In this study, we tested our hypothesis by comparing the effects of direct exposure of human disc cells to tobacco smoke condensate and nicotine on cell viability and metabolic activity. We showed that smoke condensate, which contains all of the water-soluble compounds inhaled by smokers, exerts greater detrimental effects on human disc cell viability and metabolism than nicotine. Smoke condensate greatly induced an inflammatory response and gene expression of metalloproteinases while reduced active matrix synthesis and expression of matrix structural genes. Therefore, we have demonstrated that disc cell exposure to the constituents of tobacco smoke has negative consequences which have the potential to alter disc matrix homeostasis. ß

Study Design NF-κB activity was pharmacologically and genetically blocked in an accelerated aging mouse model to mitigate age-related disc degenerative changes. Objective To study the mediatory role of NF-κB signaling pathway in age-dependent intervertebral disc degeneration. Summary of Background Data Aging is a major contributor to intervertebral disc degeneration (IDD), but the molecular mechanism behind this process is poorly understood. NF-κB is a family of transcription factors which play a central role in mediating cellular response to damage, stress, and inflammation. Growing evidence implicates chronic NF-κB activation as a culprit in many aging-related diseases, but its role in aging-related IDD has not been adequately explored. We studied the effects of NF-κB inhibition on IDD using a DNA repair-deficient mouse model of accelerated aging (Ercc1-/Δ mice) previously been reported to exhibit age-related IDD. Methods Systemic inhibition of NF-κB activation was achieved either genetically by deletion of one allele of the NF-κB subunit p65 (Ercc1-/Δp65+/- mice) or pharmacologically by chronic intra-peritoneal administration of the Nemo Binding Domain (8K-NBD) peptide to block the formation of the upstream activator of NF-κB, IκB Inducible Kinase (IKK), in Ercc1-/Δ mice. Disc cellularity, total proteoglycan content and proteoglycan synthesis of treated mice and untreated controls were assessed. Results Decreased disc matrix proteoglycan content, a hallmark feature of IDD, and elevated disc NF-κB activity were observed in discs of progeroid Ercc1-/Δ mice and naturally aged wild-type compared to young WT mice. Systemic inhibition of NF-κB by the 8K-NBD peptide in Ercc1-/Δ mice increased disc proteoglycan synthesis and ameriolated loss disc cellularity and matrix proteoglycan. These results were confirmed genetically by using the p65 haploinsufficient Ercc1-/Δp65+/- mice. Conclusion These findings demonstrate that the IKK/NF-κB signaling pathway is a key mediator of age-dependent IDD and represents a therapeutic target for mitigating disc degenerative diseases associated with aging.