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

Abstract

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.

Off-the-Shelf Nipple Engineering

“…7). Safranin-O staining of explanted xenograft cartilage revealed depletion of GAG content as expected after decellularization 8,9 with persistent preservation of its collagen-containing extracellular matrix after 6 months in vivo (Fig. 8).…”

“…The decision to use decellularized ovine costal cartilage as a proxy biomaterial for decellularized allograft was made because of the expense of commercially decellularized human allograft, which lead to our development of a protocol for the economical production of large quantities of acellular cartilage. 8,9 As was the case with vital cartilage within an immunocompetent host, the scaffolded cartilage particulate infill became invested with host tissue, resulting in a time-dependent increase in volume within the scaffold. Although a moderate inflammatory infiltrate was seen after 1 month in vivo, by 3 months, it had lessened significantly, and by 6 months, it had subsided, and the cartilage macroarchitecture appeared intact.…”

“…Given the previously reported success demonstrated by implantation of scaffolded viable processed autologous cartilage in an athymic rodent model, this study sought to determine the extent to which decellularized cartilage, which could be obtained “off the shelf,” might produce similar results in an immunocompetent host. The decision to use decellularized ovine costal cartilage as a proxy biomaterial for decellularized allograft was made because of the expense of commercially decellularized human allograft, which lead to our development of a protocol for the economical production of large quantities of acellular cartilage 8,9 …”

“…All floating ribs were isolated and minced with a #10 blade scalpel into 1-mm 3 cubes or zested into flakes (1 × 1 mm [length × width], 0.2-mm thickness and volume of approximately 0.2 mm 3 ) using a grater. Processed cartilage was decellularized as previously described 8,9 …”

“…Several racks of lamb (<24 hours after slaughter) were purchased from a local butcher and prepared for decellularization as previously described by our laboratory. 8,9 All floating ribs were isolated and minced with a #10 blade scalpel into 1-mm 3 cubes or zested into flakes (1  1 mm [length  width], 0.2-mm thickness and volume of approximately 0.2 mm 3 ) using a grater. Processed cartilage was decellularized as previously described.…”