Enhanced decellularization technique of porcine dermal ECM for tissue engineering applications

Abstract

Effective removal of cellular components while retaining extracellular matrix (ECM) proteins is the ultimate goal of decellularization. The aim of this study is to produce a decellularized ECM with highly preserved ECM proteins and to determine the effect of isopropanol as a decellularization solvent on the characteristics of the decellularized porcine skin. Two different protocols were used for porcine skin decellularization. Protocol 1 consisted of Triton-X and sodium dodecyl sulfate (SDS) in water while protocol 2 consisted of Triton-X and SDS in 70% isopropanol. After decellularization, DNA components decreased significantly in protocol 2 with lower amount of lipid content and higher ECM proteins such as collagen (92.91 ± 9.02 μg/mg sample), α-elastin (142.32 ± 6.74 μg/mg sample) and sulfated glycosaminoglycan (sGAG; 7.44 ± 1.30 μg/mg sample) compared with protocol 1 ECM. Higher amount of vascular endothelial growth factor (VEGF; 11.26 ± 0.44 pg/mg sample) content was quantified in protocol 2 compared with protocol 1 while higher trace amount of bone morphogenic protein 2 (BMP-2; 0.28 ± 0.04 pg/mg sample) was also observed in protocol 2 compared with protocol 1. Protocol 2 ECM did not significantly affect the cell viability and exhibited no cytotoxicity when exposed to three different cell lines: L929 fibroblast cells, MC3T3-E1 pre-osteoblast cells, and rat mesenchymal stem cells (BMSC). Subcutaneous implantation after 7 and 21 days revealed higher cell infiltration in protocol 2 ECM and enhanced neovascularization. Isopropanol/surfactants proved to be effective in cell and lipid removal during decellularization while preserving the higher amount of ECM proteins.

Keywords: Decellularization; Extracellular matrix; Isopropanol; Porcine skin.