In this study, we sought to explore an efficient decellularization protocol for bovine pericardia with better extracellular matrix preservation and good biocompatibility. Bovine pericardia were decellularized by sodium dodecyl sulphate (SDS), SDS + sodium deoxycholate (SD), Triton X-100 (TX), TX + SD (TS), freeze–thaw cycles + SDS + SD (FSS) and freeze–thaw cycles + TX + SD (FTS), respectively. Untreated pericardia were used as native control. Histological examination, residual cellular content analysis, biochemical and biomechanical evaluations and cytotoxicity assay were performed to investigate decellularization efficiency, xenoantigens removal, extracellular matrix preservation and biocompatibility. In vivo biocompatibility was evaluated using a subcutaneous implantation method in rats. Among these protocols, FSS and FTS protocols were the most effective methods to remove both the DNA material and the galactose-α-1,3-galactose antigen. TX, TS and FTS bovine pericardia maintained the collagen content and had no cytotoxicity to human umbilical vein endothelial cells. The contents of elastin and glycosaminoglycan were lost to different degrees after decellularization, with the highest content of preservation with TX, followed by TS and FTS. Consistently, no significant difference was found between native bovine pericardia and TX, TS or FTS bovine pericardia. In vivo, FTS implants had minimal infiltration of macrophages and T-lymphocytes, with no histological evidence of peri-implant necrosis and calcification. These results suggested that the FTS protocol showed optimal decellularization results with better extracellular matrix preservation and good biocompatibility. It may be a suitable protocol for producing a suitable scaffold for heart tissue engineering.

Decellularization is a promising technique to produce natural scaffolds for tissue engineering applications. However, non-crosslinked natural scaffolds disfavor application in cardiovascular surgery due to poor biomechanics and rapid degradation. Herein, we proposed a green strategy to crosslink and functionalize acellular scaffolds via the self-assembly of copper@tea polyphenol nanoparticles (Cu@TP NPs), and the resultant nanocomposite acellular scaffolds were named as Cu@TP-dBPs. The crosslinking degree, biomechanics, denaturation temperature and resistance to enzymatic degradation of Cu@TP-dBPs were comparable to those of glutaraldehyde crosslinked decellularized bovine pericardias (Glut-dBPs). Furthermore, Cu@TP-dBPs were biocompatible and had abilities to inhibit bacterial growth and promote the formation of capillary-like networks. Subcutaneous implantation models demonstrated that Cu@TP-dBPs were free of calcification and allowed for host cell infiltration at Day 21. Cardiac patch graft models confirmed that Cu@TP-dBP patches showed improved ingrowth of functional blood vessels and remodeling of extracellular matrix at Day 60. These results suggested that Cu@TP-dBPs not only had comparable biomechanics and biostability to Glut-dBPs, but also had several advantages over Glut-dBPs in terms of anticalcification, remodeling and integration capabilities. Particularly, they were functional patches possessing antibacterial and proangiogenic activities. These material properties and biological functions made Cu@TP-dBPs a promising functional acellular patch for cardiovascular applications.