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Objective Hyaluronic acid–transglutaminase (HA-TG) is an enzymatically crosslinkable adhesive hydrogel with chondrogenic properties demonstrated in vitro and in an ectopic mouse model. In this study, we investigated the feasibility of using HA-TG in a collagen scaffold to treat chondral lesions in an ovine model, to evaluate cartilage regeneration in a mechanically and biologically challenging joint environment, and the influence of the surgical procedure on the repair process. Design Chondral defects of 6-mm diameter were created in the stifle joint of skeletally mature sheep. In a 3-month study, 6 defects were treated with HA-TG in a collagen scaffold to test the stability and biocompatibility of the defect filling. In a 6-month study, 6 sheep had 12 defects treated with HA-TG and collagen and 2 sheep had 4 untreated defects. Histologically observed quality of repair tissue and adjacent cartilage was semiquantitatively assessed. Results HA-TG adhered to the native tissue and did not cause any detectable negative reaction in the surrounding tissue. HA-TG in a collagen scaffold supported infiltration and chondrogenic differentiation of mesenchymal cells, which migrated from the subchondral bone through the calcified cartilage layer. Additionally, HA-TG and collagen treatment led to better adjacent cartilage preservation compared with empty defects ( P < 0.05). Conclusions This study demonstrates that the adhesive HA-TG hydrogel in a collagen scaffold shows good biocompatibility, supports in situ cartilage regeneration and preserves the surrounding cartilage. This proof-of-concept study shows the potential of this approach, which should be further considered in the treatment of cartilage lesions using a single-step procedure.