Numerous cartilage repair procedures have been developed for focal lesions to minimize suffering and possibly prevent the development of osteoarthritis with a focus on so-called one-step procedures. The aim of this work was to investigate the effects of both focal cartilage defects and a biomaterial (ChondroFiller) on the corresponding articular cartilage. On a friction test stand, 18 porcine osteochondral cylinders were tested in six experimental setups under cyclic loading (33 N) against a friction partner in saline solution. The friction partner (cartilage, bone, cartilage defect, cartilage defect with ChondroFiller) and the running times (1 hour and 6 hours) were varied. The damage to the osteochondral cylinders was assessed histologically using a visual damage classification. The cartilage versus bone group showed severe cartilage damage in both the one-hour and six-hour experiments, with an average damage score of 3.5. Damage in the cartilage versus cartilage defect group was moderate, with damage values of 2.5 (1 h) and 2.67 (6 h). The cartilage versus cartilage defect with ChondroFiller group showed a damage value of 2.67 for the one-hour and 2.5 for the six-hour trials. Even focal grade IV cartilage lesions can lead to significant damage to the corresponding cartilage in vitro. The damage could not be reduced by the use of ChondroFiller, likely because of the initial instability of this biomaterial. Therefore, a biomaterial must be stable in the beginning with regard to full weight-bearing, or joint loading should be delayed until stable filling of the defect is achieved.