(80c) A  Novel Supercritical CO2-Based Treatment for Decellularization That Maintains Mechanical and Structural Integrity

We have previously presented a novel, hybrid process for decellularizing a porcine aorta, while maintaining structural and mechanical integrity. The process comprises brief exposure to an aqueous solution (for cell disruption), following by washing with supercritical CO₂(to remove cellular debris and aqueous residue). In addition to achieving decellularization, we now demonstrate that the aorta so treated maintains favorable mechanical properties, and that the structural proteins collagen and elastin maintain structure that is much closer to the native tissue than aorta treated with a standard decellularization method.

Whether derived from synthetic or natural materials, TE scaffolds must be sterile, porous, mechanically strong, biocompatible, bioactive, and properly direct cell proliferation and differentiation. Additionally, the scaffold fabrication process and subsequent treatments may introduce several structural and biochemical deficiencies, including loss of mechanical strength, loss of surface activity, denaturation of extracellular matrix (ECM) proteins, scaffold dehydration, and residual cytotoxicity of some solvents, detergents, and/or crosslinking agents. All of these challenges require continual development of novel and innovative scaffold fabrication methods. In 2016, our lab presented a novel hybrid decellularization method that utilizes supercritical carbon dioxide (scCO₂) and sodium dodecyl sulfate (SDS) to decellularize a tissue. We demonstrated that the hybrid decellularization method was able to completely decellularize porcine aorta faster than a standard SDS method.

In this work, scaffolds of porcine aorta were treated with different methods: a standard aqueous method using surfactant solutions (control), a SC CO2 method using ethanol as an additive, and the hybrid of aqueous treatment followed by CO2 rinsing. The mechanical properties of decellularized scaffolds were evaluated by performing a uniaxial ring test. Arterial tissue is non-Hookean and does not have a linear modulus of elasticity, but contributions to modulus of elasticity from elastin (E_elastin, 0-8% strain) and collagen (E_collagen, 12-20% strain) can be determined from stress-strain curves. These values were calculated for the native tissue, hybrid treatment, and treatments with only SDS and only scCO_2. . The hybrid treatment best maintained the mechanical properties of the scaffold, as substantial protein denaturing was observed during SDS treatment and excessive dehydration was observed during treatment with only scCO₂and ethanol.

The microscopic arrangement of ECM fibers (elastin and collagen) was visualized by staining with Masson’s trichrome. For the hybrid treatment, complete decellularization is observed along with a distribution of collagen that closely resembles the native aorta. SDS treatment also removes cells, but disruption of collagen is observed.

Finally, a methylene blue assay was used to quantify residual SDS for the standard SDS and hybrid SDS/scCO₂ decellularization protocols. Just one hour of treatment with scCO₂ removed a similar amount of SDS compared with a whole day of washing with phosphate-buffered saline. Prolonging scCO₂treatment beyond one hour would likely reduce residual SDS concentration below the cytotoxic level of 0.002%.

Overall, scaffolds produced by the hybrid SDS/scCO₂ decellularization protocol have more desirable mechanical and biochemical properties compared to standard treatments.