Comparison of detergent-based decellularization protocols for the removal of antigenic cellular components in porcine aortic valve.

Abstract

Various detergent-based protocols are used to remove cells and cellular debris in porcine aortic valve (PAV). However, the removal of antigenic cellular components has not been thoroughly elucidated to date. In this study, we used 4 detergent-based protocols to decellularize PAVs and aimed to evaluate their effects on removing antigenic cellular components. Porcine aortic valves were decellularized using sodium dodecyl sulfate (SDS), SDS in combination with sodium deoxycholate (SDS/SD), Triton X-100, and Triton X-100 in combination with SD (Triton X-100/SD), respectively. Untreated PAVs were used as controls. Immunohistochemical and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analyses were performed to determine the removal of antigenic protein components. Histological, biochemical, and biomechanical analyses were performed to determine the preservation and mechanical properties of the extracellular matrix. PAV tissues were implanted subcutaneously in Sprague Dawley rats to evaluate the host immune response. Implanted PAVs were taken out at the indicated time points for histological and immunohistochemical examinations. All 4 protocols effectively removed the membrane antigenic proteins major histocompatibility complex I molecule and galactose-α-1,3 galactose. The SDS/SD protocol was the most effective method to remove the cytoplasmic cytoskeletal proteins, vimentin and α-SMA, and SDS alone partly removed vimentin protein. The SDS/SD protocol was the most effective method to remove nuclear DNA with residual DNA below 50ng/mg, followed by the SDS protocol with residual DNA of 74.9ng/mg. The SDS protocol was the most effective method to remove proteins ranged from 10 to 55kDa, followed by the SDS/SD protocol. SDS and SDS/SD PAV implants attracted fewer neutrophils in vivo on postoperative day 3. The infiltration of macrophages and T lymphocytes was significantly lower in SDS and SDS/SD implants on days 14 and day 28. All of the decellularized protocols that were examined greatly reduced the contents of collagen, elastin, and glycosaminoglycan compared to controls. Biomechanical analysis revealed significant differences in ultimate tensile strength and Young's modulus between control PAVs and decellularized PAVs generated using SDS, SDS/SD, Triton X-100, or Triton X-100/SD. These results indicated that SDS-based protocols more effectively removed antigenic cellular components compared to Triton X-100-based protocols. These results are clinically significant because complete removal of antigenic determinants is critical to decrease adverse immune-mediated and inflammatory responses to a PAV when used in xenogeneic application.