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To study the relationship between 12-month leisure-time physical activity (LTPA) level and changes in estimated biochemical composition of tibiofemoral cartilage in postmenopausal women with mild knee osteoarthritis (OA).Originally, 87 volunteer postmenopausal women, age 60 to 68 yr, with mild knee OA (Kellgren Lawrence I/II and knee pain) participated in a randomized controlled, 4-month aquatic training trial (RCT), after which 76 completed the 12-month postintervention follow-up period. Self-reported LTPA was collected along the 12-month period using a diary from which MET task hours per month were calculated. Participants were divided into MET task hour tertiles: 1, lowest (n = 25); 2 = middle (n = 25) and 3 = highest (n = 26). The biochemical composition of the cartilage was estimated using transverse relaxation time (T2) mapping sensitive to the properties of the collagen network and delayed gadolinium-enhanced magnetic resonance imaging of the cartilage (dGEMRIC index) sensitive to the cartilage glycosaminoglycan content. Secondary outcomes were cardiorespiratory fitness, isometric knee extension and flexion force, and the knee injury and OA outcome questionnaire.During the 12-month follow-up period, there was a significant linear relationship between higher LTPA level and increased dGEMRIC index changes in the posterior region of interest (ROI) of the lateral (P = 0.003 for linearity) and medial (P = 0.006) femoral cartilage. Furthermore, these changes were seen in the posterior lateral femoral cartilage superficial (P = 0.004) and deep (P = 0.007) ROI and in the posterior medial superficial ROI (P < 0.001). There was no linear relationship between LTPA level and other measured variables.These results suggest that higher LTPA level is related to regional increases in estimated glycosaminoglycan content of tibiofemoral cartilage in postmenopausal women with mild knee OA as measured with dGEMRIC index during a 12-month period.