Treatment of osteochondral injuries is challenging, and no gold standard has been established. Layered cell-free scaffolds are a new treatment option for these defects. The aim of this study was to evaluate the osteochondral repair in patients treated with the MaioRegen(®) scaffold, a cell-free biomimetic scaffold consisting of type I collagen and hydroxyapatite. Treatment using this scaffold has previously shown promising clinical results. Ten patients with osteochondral lesions in the knee (n=6) or in the talus (n=4) were enrolled. The patients underwent pre-operative MRI and CT scans and were assessed at 1- and 2.5-year timescales post-operatively. The cartilage and bone formations were evaluated semi-quantitatively using the MOCART score. Knee patients were clinically evaluated using KOOS, subjective IKDC and Tegner scores, whereas ankle patients were evaluated using AOFAS Hindfoot and Tegner scores. Two patients were re-operated and excluded from further follow-up due to treatment failure. None of the patients had complete regeneration of the subchondral bone evaluated using CT. At 2.5years, 6/8 patients had no or very limited (<10%) bone formation in the defects and 2/8 had 50-75% bone formation in the treated defect. MRI showed no improvement in the MOCART score at any time point. The IKDC score improved from 41.3 to 80.7, and the KOOS pain subscale improved from 63.8 to 90.8 at 2.5-year follow-up. No improvement was found with the remaining KOOS subscales, the Tegner or AOFAS Ankle-Hindfoot score. Treatment of osteochondral defects in the ankle and knee joint with a biomimetic scaffold resulted in incomplete cartilage repair and poor subchondral bone repair at 1- and 2.5-year follow-up. Clinical significant improvements were observed. These results raise serious concerns about the biological repair potential of the MaioRegen(®) scaffold, and we advise to use the MaioRegen(®) scaffold with caution. Prospective therapeutic study, Level IV.