Production of a Low-Cost, Off-the-Shelf, Decellularized Cartilage Xenograft for Tissue Regeneration

BACKGROUND: Reconstruction of cartilaginous deformities is a well-established surgical challenge with high levels of unpredictability and complication. Because of the morbidity associated with autologous cartilage grafting, combined with its limited supply and the significant expense of commercially decellularized allografts, increasing efforts have sought to produce an acellular, nonimmunogenic cartilage xenograft. We have developed and validated a novel protocol for high throughput decellularization of ovine costal cartilage with immediate translational potential for pre-clinical investigation of novel strategies for cartilaginous reconstruction. METHODS: Floating ribs were isolated from freshly slaughtered rack of lamb and after cleaning, the ribs were either minced into 2 mm cubes or zested into 1 mm flakes. Tissue was then decellularized via a protocol consisting of four freeze/thaw cycles, digestion with trypsin, incubation in hyperosmolar and hypo-osmolar salt solutions, with incubation in 1% Tween following both the hyperosmolar and hypo-osmolar steps, a 48-hour incubation in nucleases, DNA elution via EDTA, and two terminal sterilization steps. Protocol success was evaluated via histologic analysis with H&E, DAPI, and safranin-O staining, as well as DNA quantification. RESULTS: Histologic analysis of the decellularized tissue revealed a significant reduction in nuclei as evidenced by H&E and DAPI staining (p