Epoxy Chitosan-Crosslinked Acellular Bovine Pericardium with Improved Anti-calcification and Biological Properties

Glutaraldehyde (GA) was conventionally used to crosslink bovine pericardium to prepare bioprosthetic heart valves (BHVs), which usually fail within 10 years because of valve deterioration and calcification. To overcome the high cytotoxicity and severe calcification of GA-crosslinked BHVs, a quaternary ammonium salt of epoxy chitosan (epoxy group-modified 3-chlorine-2-hydroxypropyl trimethyl chitosan, abbreviated as "eHTCC") was developed to modify the acellular bovine pericardium to substitute GA and improve its anti-calcification and biocompatible properties. Mechanical test, enzymatic stability test, blood compatibility assay, and cytocompatibility assay were used to investigate its mechanical property and biocompatibility. The anti-calcification effect of the eHTCC-modified bovine pericardium (eHTCC-BP) was assessed by in vitro assay and rat subcutaneous implantation assay. The results showed that eHTCC-BP could improve the mechanical properties and anti-enzymolysis ability of BP, as well as retain the original three-dimensional structure, compared with the uncrosslinked-BP group. Moreover, the in vivo calcification level of the eHTCC-BP group was much lower than that of the GA-BP group, which was 5.1% (2 weeks), 2.3% (4 weeks), and 0.8% (8 weeks) of the GA-BP group. In summary, this study demonstrated that eHTCC could be a potential crosslinking agent for the extracellular matrix for its favorable crosslinking effects, anti-enzymolysis, anti-calcification, and biocompatibility.