Thirty six subjects, between the ages of 47 and 75, were included in the analysis. Eighteen subjects presented with current knee pain, endorsed experiencing chronic pain, were confirmed to have radiographic knee osteoarthritic changes , and were without rheumatoid arthritis, heart disease or uncontrolled medical conditions including high blood pressure, diabetes, and gout. The no pain group was comprised of 18 individuals presenting without chronic knee pain or medical comorbidities. The research was conducted on the Clinical Research Unit at the University of Florida (UF). The protocol and procedures were approved by UF's IRB and informed consent was obtained for each participant. Methods described are limited to those relevant to the current analyses.
Subjects completed a health history questionnaire, the Graded Chronic Pain Scale (GCPS) , the Perceived Stress Scale, 10 item (PSS) , the Center for Epidemiologic Studies Depression scale (CES-D) , and a physical exam including knee radiographs. Additionally, duration of knee pain in months was collected. Baseline blood samples were collected and peripheral blood mononuclear cells (PBMCs) were isolated to assess TL. Genomic DNA was extracted from lymphocytes in whole blood using a commercially available kit (Qiagen Flexigene DNA Kit, Qiagen, Valencia, CA). DNA samples were quantified, normalized and plated in 96-well plates. Relative telomere length was measured by quantitative real-time polymerase chain reaction (qPCR). Each qPCR experiment contained telomere primers and β2-globin (control gene) primers .
Telomeres and a single copy gene (β2-globin) were amplified in all the samples in triplicate on each plate. The ΔΔCt method was performed using SDS V.2.1 software from Applied Biosystems. Ct values from each sample were then used to calculate the ratio of telomere to single copy gene (T/S) values using the formula (2 Ct
β2-globin)-1. Relative T/S values were calculated by the formula 2 -ΔΔCt. Using this formula, we determined the TL of each sample relative to the mean T/S for all samples .
Independent sample t-tests, ANOVA, and Chi Square or Fisher's Exact tests were implemented for between group comparisons of demographic variables as appropriate. Due to the limited sample size, covariates were selected based on the following criteria: 1) previous association of the measure with TL, 2) association with TL in the current study, 3) not significantly correlated with other covariates, and 4) relevance to the study design. Based on previous findings, potential covariates considered included age, sex, waist/hip ratio (WHR), BMI, annual income, education, exercise frequency, smoking status, and depression. A median PSS split was used to categorize low stress (< 15) and high stress (≥ 15) as previously reported . Four groups were developed based on pain status, no pain or chronic pain, and PSS total scores and were categorized as no pain/low stress (NPLS); no pain/high stress (NPHS); chronic pain/low stress (CPLS); and chronic pain/high stress (CPHS). General Linear Models (GLM) were implemented to analyze TL data. All analyses were completed with IBM SPSS Statistics 19.