Osteochondral defect model and treatment

Ten-week-old (~ 250 g) male Sprague–Dawley rats were anesthetized using Zoletil/Rompun (50 mg/kg and 5–10 mg/kg, respectively) administered by intraperitoneal (IP) injection in an IACUC-approved animal laboratory facility. After shaving the right knee of each rat and sterilizing it using povidone-iodine swabs, a medial parapatellar incision was made on the knee, and the patella was deflected laterally to expose the trochlear surface. A hole 1.5 mm in diameter and 1 mm deep was drilled into the center of the trochlea using a High-Speed Rotary Micromotor Kit (Seashin, Daegu, Korea) with a 1.5 mm trephine (Fine Science Tools, Foster City, CA, USA) to induce an osteochondral defect. The experimental animals were divided randomly into four groups: the normal group (n = 5), the collagen treatment group (n = 5), the collagen with hNCs treatment group (n = 5) and the collagen with hNCs-3D treatment group (n = 5). The animals were housed in pairs for the duration of the experiment. After defect creation, hNCs-2D or hNCs-3D encapsulated in collagen was implanted by injection using a 1 ml syringe (BD Biosciences, Franklin Lakes, NJ, USA) to achieve full defect filling. The gel usually hardens within 10 min, and hNCs-2D or hNCs-3D encapsulated in collagen was fitted in the defect site. At 4 and 8 weeks, the animals were double euthanized using CO₂ gas and cervical dislocation, and the knees were harvested. The samples were fixed in 10% formalin for 24 h for analysis. The left knees (uninjured) were also harvested as animal-specific internal controls for observation.