Abstract:
Although the adverse effects of smoking for human musculoskeletal system have been well accepted, less attention has been paid by researchers to the relevance of cigarette smoke to the onset of osteoarthritis (OA). Here, we investigated the effects of cigarette smoke extract (CSE) on human primary chondrocyte function. In addition, we investigated whether the pharmacologic treatment of dexamethasone was beneficial to chondrocytes impaired by CSE, and if not, whether it could be substituted by other treatments, such as acetaminophen and NSAIDs. Finally, we evaluated the effects of hyaluronic acid (HA) and HA combinatory treatments (dexamethasone, acetaminophen or diclofenac) on the chondrocytes impaired by CSE, in order to determine a potential therapeutic alternative for clinical application to smokers undergoing symptomatic OA.
All human tissues were obtained in accordance with the ethical approval of the University Hospital Tübingen and with patients’ written consent. Human primary chondrocytes were exposed to increasing concentrations (0%, 0.1%, 0.5%, 1%, 5%, 10%) of CSE (containing 3.6 ng/ml to 72 ng/ml nicotine and 40 ng/ml to 800 ng/ml tar). Cell viability was analyzed by resazurin conversion assay and SRB staining, matrix formation was stained using Alcian blue and Safranin-O staining. The generation of free radical was evaluated by DCFH-DA assay. Semi-quantitative RT-PCR was performed to analyze gene expressions.
Our present study demonstrated that the mitochondrial activity, total protein content and the accumulation of matrix were dose- and time-dependently inhibited by CSE in primary human chondrocytes. Moreover, increased oxidative stress led to cell death by 10% CSE, which is associated with approximately smoking one pack a day. As an anti-inflammatory treatment strategy, traditional pharmacologic therapy with dexamethasone (Dex) was evaluated. Clinical doses of Dex were toxic to the cells, and long-time incubation with lower doses (4−400 μg/ml) of Dex would lead to a hypertrophic chondrocyte phenotype. To substitute dexamethasone, a clinical dosage of diclofenac (Dic) and acetaminophen (Ace) was tested on chondrocytes. Interestingly, therapeutic doses of Dic (1 μg/ml) and Ace (10 μg/ml) did not augment the detrimental effects of CSE on the overall metabolisms of chondrocytes. Additionally, a clinical dose of HA (5 mg/ml) and/or HA combined with Dic, Ace, or doses of Dex had protective effects on the CSE-exposed chondrocytes, as they significantly inhibited or trap the generation of free radical and promoted the viability and ECM accumulation of cells. Our study demonstrates that cigarette smoke induces cell death through elevating oxidative stress and demolishes cartilage formation. Intra-articular (IA) injection of HA combined with therapeutic doses of analgesic/anti-inflammatory agents (Ace or Dic) could reverse the detrimental effects of CSE on primary human chondrocytes, thus opening up potential therapeutic alternatives in treating smokers to suffering from symptomatic OA.
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