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Sustainable And Efficacious Sterilization And Decellularization Of Xenograft Cartilage Using Supercritical Carbon Dioxide
Nicholas Andrew Vernice, A.B.1, Sabrina Shih, B.A.1, Carly Askinas, B.S.1, Xue Dong, M.D., Ph.D.1, David M. Bednarski, M.S.2, Tony Eisenhut, B.S.2, Jason A. Spector, M.D.1.
1Weill Cornell Medicine, New York, NY, USA, 2NovaSterilis, Inc., Lansing, NY, USA.

PURPOSE: Current decellularization techniques have important limitations, including obligatory use of harsh ionized substances or detergents that compromise the physical integrity of the resulting extracellular matrix (ECM) scaffold and, consequently, its biological performance. Furthermore, the detergents used are increasingly recognized to be harmful to the environment and may soon restricted from commercial use. Supercritical CO2 (scCO2) is an appealing alternative, applying the principles behind supercritical fluid extraction and sterilization to the removal of cellular components. In this work, we evaluate the efficacy of scCO2 in both sterilization and decellularization of cartilage xenograft in order to develop a highly efficacious, environmentally conscious, and biocompatible alternative to currently available technologies.
METHODS: Xenograft preparation: Racks of lamb were purchased from a local butcher. Floating ribs were minced into ∼8 mm3 cubes or zested into flakes (1 mm × 1 mm). Samples were subjected to: 1) sterilization; 2) sterilization and decellularization; or 3) sterilization, decellularization, and a pretreatment wash. Sterilization: Samples were placed in a NovaSterilis™ Nova2200 unit and subject to standard NovaSterilis™ sterilization parameters, including temperature (35 °C), NovaKill solution (16 mL), scCO2 dwell time (90 min.), and pressure (>1,200 psi). All samples were plated to allow for enumeration of bioburden reduction. At least 3 samples with biologic indicators were placed within the packaging and processed with each run. Decellularization: Samples were subject to the standard parameters of the Nova2200 system as previously described by NovaSterilis™ with an ethanol to scCO2 volume ratio of 1:3.3. Wash: Samples were serially soaked in saline and agitated with exposure to 16 mL 3% H2O2 and a 30-minute scCO2 run at 35 °C and 1,436 psi. Histology: After treatment, hematoxylin and eosin (H&E), DAPI, and safranin-O staining were performed. DNA Quantification: DNA content was quantified in raw and decellularized graft samples using the DNeasy Blood & Tissue kit (Qiagen Inc.) with a DNA content <50 ng DNA/mg tissue considered decellularized as per the literature standard.
RESULTS: No microbial growth was detected after 14 days incubation. H&E staining revealed preservation of tissue architecture after both sterilization and decellularization. DAPI staining demonstrated depletion of nuclei in the decellularized zested samples, but persistence of visible nuclei in the minced samples (Figure 1). Safranin-O staining revealed GAG depletion after decellularization. DNA content in raw minced and zested samples was 192.2 ng DNA/mg tissue and 321.6 ng DNA/mg tissue, respectively, while DNA content in sterilized and decellularized minced and zested samples were 24.8 ng DNA/mg tissue and 11.6 ng DNA/mg tissue, respectively (industry standard requirement for decellularization <50 ng DNA/mg tissue); after sterilization, decellularization, and the chemical wash, minced and zested samples contained 17.6 ng DNA/mg tissue and 4 ng DNA/mg tissue respectively.
CONCLUSIONS: These preliminary data suggest that scCO2 sufficiently sterilizes and decellularizes ovine xenograft, thereby supporting scCO2 as an efficacious, commercially appealing, and ecologically responsible alternative to current decellularization strategies.


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