Establishment and Evaluation of a Sheep Model of Full-thickness Osteochondral Defect

Cartilage plays a vital role in preserving the mechanical competence of the skeletal system by providing a smooth and lubricated surface in the diarthrodial joints, avoiding erosion of articular surface. Despite its remarkable ability to support and distribute physical and mechanical loads in the joints, in the case of injury, articular cartilage has a limited capacity for intrinsic healing and repair. The accumulation of strain in the articular cartilage can lead to the pathological condition known as osteoarthritis, the most common form of degenerative joint disease in the world, which affects almost 10 to 12%of the adult population worldwide.

Cartilage regeneration and the restoration of function in arthritic joints represent significant advances in regenerative medicine. Promising approach include the use of autologous chondrocytes and stem cells in combination with biomaterials. To validate these strategies, suitable animal models are essential, while smaller animals such as rats and rabbits are useful for preliminary studies, large animals like sheep are more appropriate due to their anatomical and biomechanical similarities to human joints.

Next, we present an efficient protocol for inducing osteochondral defects in large animals, which provides an ideal model for regenerative medicine research. After disinfecting the shaved knee in the surgery room, place the animal in dorsal decubitus position. To define the exact location for the surgical cut, touch the knee of the animal to find the articular cavity.

Perform the surgical cutting with approximate size of three to five centimeters. Make sure that the incision is being made in the region of the animal's condyle. Remove the tissue layers underlying the skin to access the condyle.

Keep the incision area clean to avoid potential contamination, and ensure a good visibility for the procedure. Perform the collection of synovial fluid by using a syringe and needle. Position the retractors to fully expose the condylar surface.

The more exposed the condyle is, the easier it will be to induce injury in the next step. Use an orthopedic drill attached to an eight millimeter trephine drill to promote an injury to the animal's condyle. Before initiating to drill, make sure that the trephine is properly positioned in the center of the condyle to avoid injuries of undesired dimensions.

Use the orthopedic hammer and osteotome to perform the surgical extraction of the osteochondral plug. Clean the wound extension with sterile gauze, ensuring the inside of the lesion is free of blood and tissue debris. Carefully apply 200 microliters of the liquid gelMA formulation to the inside of the injury until it reaches the surface.

Direct the UV light to the injury for three minutes at a distance of approximately five centimeters, allowing gelMA cross-linking. Carefully remove the retractors. Close the lesion using independent sutures for each tissue layer to avoid the formation of seroma.

The number of sutures to be given will depend on the size of the surgical cut. Place a bandage on the sutures to avoid infections. After seven days of surgical procedure, the sheep will still have locomotion difficulties.

These difficulties are evidenced by the rotational movement observed during the walking evaluations of the animal. This observation reveals that our protocol was effective in inducing osteochondral injury in the animal's joint. Over four days, depending on the treatment given to the lesion, the animal's gait movement is reestablished and it starts to walk correctly.

Histological evaluation with hematoxylin and eosin staining was performed six months after the surgical procedure. The lesions appeared as complete vertical clefts corresponding to score four on the Osteoarthritis Research Society International grading scale. In some specimens, mild synovial hyperplasia and replacement of cartilage by fibroconnective tissue were observed.

Histological scores showed substantial variability in both the natural progression and gelMA treated groups and statistical analysis revealed no significant difference between treatments. Since this is an animal experiment combined with the invasive surgical procedure, the animals may show variations in behavior and recovery. Our work demonstrated an efficient protocol for the induction of osteochondral injuries in the large animals without onset of long-term bacterial infections.