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 preclinical 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 4 freeze/thaw cycles, digestion with trypsin, incubation in hyperosmolar and hypoosmolar salt solutions, with incubation in 1% Tween following both the hyperosmolar and hypoosmolar steps, a 48-hour incubation in nucleases, DNA elution via EDTA, and 2 terminal sterilization steps. Protocol success was evaluated via histologic analysis with hematoxylin and eosin, 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 hematoxylin and eosin and DAPI staining (P < 0.01). Safranin-O staining demonstrated a depletion of glycosaminoglycan content in the decellularized cartilage but with preservation of tissue architecture. Unprocessed lamb cartilage contained 421 ± 60 ng DNA/mg of lyophilized tissue, whereas decellularized zested and minced costal cartilage contained 27 ± 2 ng DNA/mg lyophilized tissue (P < 0.0001) and 24 ± 2.3 ng DNA/mg lyophilized tissue (p < 0.0001), respectively, well below the threshold of 50 ng accepted as evidence of suitable decellularization. In comparison, commercial allograft cartilage contained 17 ± 5 ng DNA/mg of lyophilized tissue. CONCLUSIONS: We have developed a novel protocol for the decellularization of xenogeneic cartilage graft. This structurally stable, low immunogenicity decellularized cartilage can be produced at low cost in large quantities for use in preclinical investigation.
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 preclinical 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 4 freeze/thaw cycles, digestion with trypsin, incubation in hyperosmolar and hypoosmolar salt solutions, with incubation in 1% Tween following both the hyperosmolar and hypoosmolar steps, a 48-hour incubation in nucleases, DNA elution via EDTA, and 2 terminal sterilization steps. Protocol success was evaluated via histologic analysis with hematoxylin and eosin, 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 hematoxylin and eosin and DAPI staining (P < 0.01). Safranin-O staining demonstrated a depletion of glycosaminoglycan content in the decellularized cartilage but with preservation of tissue architecture. Unprocessed lamb cartilage contained 421 ± 60 ng DNA/mg of lyophilized tissue, whereas decellularized zested and minced costal cartilage contained 27 ± 2 ng DNA/mg lyophilized tissue (P < 0.0001) and 24 ± 2.3 ng DNA/mg lyophilized tissue (p < 0.0001), respectively, well below the threshold of 50 ng accepted as evidence of suitable decellularization. In comparison, commercial allograft cartilage contained 17 ± 5 ng DNA/mg of lyophilized tissue. CONCLUSIONS: We have developed a novel protocol for the decellularization of xenogeneic cartilage graft. This structurally stable, low immunogenicity decellularized cartilage can be produced at low cost in large quantities for use in preclinical investigation.
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