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High tibial osteotomy (HTO) is reported to be an effective treatment for varus knee osteoarthritis (OA) by redistributing the load line within the knee joint. The cell-based tissue engineering approach using mesenchymal stem cells (MSCs) has addressed the issue of articular cartilage repair in knee OA.This study aimed to compare the clinical, radiological, and second-look arthroscopic outcomes of open-wedge HTO with versus without an MSC injection and to identify the association between cartilage regeneration and HTO outcomes.Cohort study; Level of evidence, 3.Among 271 patients treated with HTO for varus knee OA from September 2009 to April 2014, patients treated with HTO alone (conventional group; n = 50) were pair-matched with those who underwent HTO with an MSC injection (injection group; n = 50) based on sex, age, and lesion size. Clinical outcomes were evaluated using the International Knee Documentation Committee (IKDC) score and Lysholm score. Radiological outcomes evaluated were the femorotibial angle and posterior tibial slope. At second-look arthroscopic surgery, cartilage regeneration was evaluated using the International Cartilage Repair Society (ICRS) grade.At the time of second-look arthroscopic surgery (mean, 12.4 months [conventional group] and 12.7 months [injection group]), the mean IKDC and Lysholm scores in each group significantly improved: conventional group, from 38.4 ± 9.2 to 55.2 ± 15.0 and from 56.7 ± 12.2 to 79.6 ± 13.5, respectively; and injection group, from 36.5 ± 4.7 to 62.7 ± 14.1 and from 55.7 ± 11.9 to 80.6 ± 15.6, respectively (P < .001 for all). Clinical outcomes at final follow-up (mean, 38.8 months [conventional group] and 37.2 months [injection group]) further improved from 62.7 ± 14.1 to 64.8 ± 13.4 (IKDC) and from 80.6 ± 15.6 to 84.7 ± 16.1 (Lysholm) (P < .001 and P = .034, respectively) only in the injection group when compared with the values at second-look arthroscopic surgery. At final follow-up, there was a significant difference in the mean IKDC and Lysholm scores between groups (P = .049 and P = .041, respectively). Overall ICRS grades, which significantly correlated with clinical outcomes, were better in the injection group than in the conventional group. Radiological outcomes at final follow-up showed improved knee joint alignment relative to patients’ preoperative conditions but showed no significant correlation with clinical outcomes or ICRS grade in either group (P > .05 for all).The group that received an MSC injection scored better on the IKDC and Lysholm scales at final follow-up than the group that did not, although these differences were relatively small. When performing HTO for patients with varus knee OA, an MSC injection should be considered as an additional procedure for improved cartilage regeneration with better clinical outcomes.