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Background: Although various factors have been identified that influence outcome after autologous chondrocyte implantation (ACI), the relevance of prior treatment of the cartilage defect and its effect concerning the outcome of second-line ACI have not been evaluated to a full extent.Hypothesis: Autologous chondrocyte implantation used as a second-line treatment after failed arthroscopic microfracturing is associated with a higher failure rate and inferior clinical results compared with ACI as a first-line treatment.Study Design: Cohort study; Level of evidence, 3.Methods: A total of 28 patients with isolated cartilage defects at the knee joint were treated with ACI after microfracture as a first-line treatment had failed (failure defined as the necessity of reintervention). These patients were assigned to group A and compared with a matched-pair cohort of patients of identical age, defect size, and defect location (group B) in which ACI was used as a first-line treatment. Failure rates in both groups were assessed. Postoperative knee status was evaluated with the International Knee Documentation Committee (IKDC) score and Knee injury and Osteoarthritis Outcome Score (KOOS), and sporting activity was assessed by use of the Activity Rating Scale. Mean follow-up times were 48.0 months (range, 15.1-75.1 months) in group A and 41.4 months (range, 15.4-83.6 months) in group B. Differences between groups A and B were analyzed by Student t test.Results: Group A had significantly greater failure rates (7 of 28 patients) in comparison with group B (1 of 28 patients; P = .0241). Mean (SD) postoperative IKDC scores revealed 58.4 (22.4) points in group A with a trend toward higher score results (69.0 [19.1] points) for patients in group B (P = .0583). Significantly different results were obtained for KOOS pain and activity of daily living subscales, whereas the remaining KOOS subscales did not show significant differences. Despite the significantly higher failure rate observed in group A, those patients did not participate in fewer activities or perform physical activity less frequently or at a lower intensity.Conclusion: Autologous chondrocyte implantation after failed microfracturing appears to be associated with a significantly higher failure rate and inferior clinical outcome when compared with ACI as a first-line treatment. [ABSTRACT FROM PUBLISHER]