Comparative Analysis of Decellularization Methods for the Production of Decellularized Umbilical Cord Matrix

Abstract

The importance of decellularized extracellular matrix (dECM) as a natural biomaterial in tissue engineering and regenerative medicine is rapidly growing. The core objective of the decellularization process is to eliminate cellular components while maximizing the preservation of the ECM’s primary structure and components. Establishing a rapid, effective, and minimally destructive decellularization technique is essential for obtaining high-quality dECM to construct regenerative organs. This study focused on human umbilical cord tissue, designing different reagent combinations for decellularization protocols while maintaining a consistent processing time. The impact of these protocols on the decellularization efficiency of human umbilical cord tissue was evaluated. The results suggested that the composite decellularization strategy utilizing trypsin/EDTA + Triton X-100 + sodium deoxycholate was the optimal approach in this study for preparing decellularized human umbilical cord dECM. After 5 h of decellularization treatment, most cellular components were eliminated, confirmed through dsDNA quantitative detection, hematoxylin and eosin (HE) staining, and DAPI staining. Meanwhile, Masson staining, periodic acid-silver methenamine (PASM) staining, periodic acid-Schiff (PAS) staining, and immunofluorescent tissue section staining results revealed that the decellularized scaffold retained extracellular matrix components, including collagen and glycosaminoglycans (GAGs). Compared to native umbilical cord tissue, electron microscopy results demonstrated that the microstructure of the extracellular matrix was well preserved after decellularization. Furthermore, Fourier-transform infrared spectroscopy (FTIR) findings indicated that the decellularization process successfully retained the main functional group structures of extracellular matrix (ECM) components. The quantitative analysis of collagen, elastin, and GAG content validated the advantages of this decellularization process in preserving and purifying ECM components. Additionally, it was confirmed that this decellularized matrix exhibited no cytotoxicity in vitro. This study achieved short-term decellularization preparation for umbilical cord tissue through a combined decellularization strategy.