From Dinner to Decell – A Novel Protocol for the Low-Cost Generation of Moldable Decellularized Xenograft Cartilage for Plastic Surgery
Ryan J. Bender, Nabih Berri, Xue Dong, Jason Harris, Sarah Caughey, Nicholas Vernice, Sabrina Shih, George Corpuz, Jason A. Spector
Laboratory of Bioregenerative Medicine & Surgery, Division of Plastic Surgery, Weill Cornell Medical College, New York, NY
Introduction: The gold standard for autologous reconstruction of cartilaginous structures in plastic surgery is patient-derived autologous cartilage, typically from the ribs, nasal septum, or bowl of the ear. While this cartilage can be used without concern for rejection, the supply is limited, and the cartilage often requires significant shaping to meet the needs of the recipient site. Cartilage has proven notoriously difficult to tissue engineer, due to the asvascularity of cartilage and the limited replication of chondrocytes. The rigidity of large cartilage pieces has led some surgeons to use a “Turkish delight” technique, mincing cartilage into small pieces that can then be molded to fit the desired shape. We seek to optimize the low-cost generation of minced and zested acellular xenograft cartilage that can be molded to fill implanted 3D-printed scaffolds and avoid rejection when implanted in immunocompetent animals.
Methods: We obtained fresh costal cartilage from 6-month old lambs and stripped them of perichondrium and surrounding tissue. The ribs were minced into 1-2 mm side length cubes or zested into flakes of cartilage <0.5 mm in thickness. The cartilage was subjected to 3 freeze-thaw cycles to disrupt cell membranes, then partially digested with dilute trypsin solution. Next, cartilage was subjected to hyperosmolar and hyperosmolar solutions to lyse cells, with Tween detergent steps in between. After DNAse/RNAse digestion to break down nucleic acids, which are highly antigenic, the tissue is rinsed and sterilized with dilute peracetic acid. Decellularization was confirmed with H&E staining, as well as DAPI staining, with DNA quantification done on lyophilized samples of tissue.
Results: All samples run through our protocol showed absent nuclei on H&E and DAPI within the minced and zested cartilage. Nuclei were sporadically noticed within calcified sections of cartilage on DAPI staining. DNA content assessment was hindered by difficulty digesting calcified sections of cartilage. Decellularized cartilage was packed into scaffolds designed with the dimensions of the human ear. These cartilage-filled scaffolds were implanted subcutaneously onto the backs of rats, with one example rat showing no signs of infection or rejection of the implants for 6 months.
Conclusion: Our protocol enables the generation of sterile, decellularized cartilage that can be packed into 3D-printed scaffolds for plastic surgery applications of cartilage tissue engineering. We are currently evaluating further optimization of the protocol, through the use of chelating agents to remove any calcification to increase efficiency of decellularization and facilitate enzymatic breakdown for DNA quantification.
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