Articular cartilage injuries are very common in orthopaedic surgery and sports medicine, and often, with time they lead to premature osteoarthritis, and consequently a decrease in quality of life and increase in costs of health care. Although mentioned procedures produce good clinical result in terms of pain relief and improvement of joint function, none of the currently used methods results in regeneration of healthy hyaline cartilage. Due to that fact, numerous preclinical and clinical studies are trying to overcome existing problems and suggest novel, improved methods to treat cartilage defects. Central hypothesis of the proposed study suggest that is possible to use adenoviral vector carrying gene for coding TGF-β1 to transduce autologous bone marrow clot which is subsequently implanted into partial-thickness cartilage defects, and the newly formed tissue within the defect is qualitatively superior to the one formed without gene therapy. The aims of the proposed study are to: evaluate translational value of the proposed method, to analyze effects of the transduction with TGF-β1 on restoration of cartilage by evaluation histological, biochemical and biomechanical parameters. Additional aim was to determine if there is a vector “leakage” from the clot into the surrounding synovial tissue. Upon obtaining local Ethical Committee approval, 28 skeletally mature sheep (1 to 3 years old) were used in this study. The sheep were randomly assigned to one of four groups. Partialthickness chondral defect was made on the weight-bearing surface of the medial condyle with standard mosaicplasty instrument, and corresponding bone marrow clots were carefully inlaid into the defects without material fixation. In the bone marrow clot (BMC) group (n=6), the sheep were implanted with untreated autologous bone marrow clot that was aspirated from iliac crest of respected animal. In the bone marrow transduced with Ad.GFP (GFP) group (n=6), the sheep were implanted with autologous bone marrow clots 96 genetically modified to over express green fluorescent protein (GFP). In the bone marrow transduced with Ad.TGF-β1 (TGF) group (n=10), the sheep were implanted with autologous bone marrow clots genetically modified to over express transforming growth factor-β1. Six untreated sheep served as a control (defect without implant). Undamaged control articular cartilage also was taken from the medial condyle of the contralateral knee joint. All sheep were euthanatized 6 months after surgery, and histological, biochemical and biomechanical parameters of the newly formed tissue from the defect were analyzed. The results confirmed the central hypothesis, as they showed that it is possible to use abbreviated ex vivo protocol for genetical modification of autologous bone marrow clots to produce repair tissue within the defect, and the use of TGF-β1 additionally improves the quality of newly formed cartilage. Histological analysis showed that new tissue formed in all the treatment groups except in the empty controls, and the biochemical analysis showed optimal content of glycosaminoglycans and collagen type II only in TGF treated group. IT AFM revealed that, on micrometer scale of tissue organization, newly formed cartilage from TGF treated group has biomechanical properties that are the closest to the native cartilage. On nanometer scale, the results are suboptimal due to high overall collagen content and low water content. PCR analysis of the synovial lining tissue could not detect residual presence of vector in none of the experimental groups. The results suggest that proposed method is a single-step procedure that can be easily implemented in standard clinical settings, and avoids the use of expensive in vitro production of autologous, engineered tissues. It was possible to produce cartilage tissue of satisfactory quality, and further improvements may be obtained with adenoviral transfer of TGF-β1 that is secreted within the clot, and it stimulates and regulates the chondrogenesis process in situ.