Articular cartilage injuries experienced at an early age can lead to the development of osteoarthritis later in life. In situ 3D printing is an exciting and innovative bio-fabrication technology that enables the surgeon to deliver tissue- engineering techniques at the time and location of need. We have created a hand- held 3D printing device (Biopen) that allows the simultaneous co-axial extrusion of bioscaffold and cultured cells directly into the cartilage defect in vivo in a single session surgery. This pilot study assesses the ability of the Biopen to repair a full thickness chondral defect and the early outcomes in cartilage regeneration, and compares these results to other treatments in a large animal model. A standardised critical-sized full thickness chondral defect was created in the weight-bearing surface of the lateral and medial condyles of both femurs of 6 sheep. Each defect was treated with one of the following treatments: (i) hand- held in situ 3D printed bioscaffold using the Biopen (HH group), (ii) pre- constructed bench-based printed bioscaffolds (BB group), (iii) micro-fractures (MF group) or (iv) untreated (Control, C group). At 8 weeks after surgery, macroscopic, microscopic and biomechanical tests were performed. Surgical 3D bio-printing was performed in all animals without any intra- or post- operative complication. The HH Biopen allowed early cartilage regeneration. Results of this study show that real-time, in vivo bioprinting with cells and scaffold is a feasible means of delivering a regenerative medicine strategy in a large animal model to regenerate articular cartilage.