Mitigation of calcification and cytotoxicity of a glutaraldehyde-preserved bovine pericardial matrix: improved biocompatibility after extended implantation in the subcutaneous rat model.

Abstract

Implanted non-crosslinked tissues suffer rapid degeneration, shrinkage and absorption, whereas standard crosslinked tissues cause local cytotoxicity and calcification. Both approaches diminish implant capacity for long-term function. The study aim was to examine the tissue-engineered characteristics (cytotoxicity, calcification potential, biocompatibility) of bovine pericardium, crosslinked with a low concentration of glutaraldehyde (GA) followed by ADAPT anti-mineralization, following prolonged implantation in a subcutaneous rat model. Bovine pericardium was decellularized with Triton X-100, deoxycholate, IgePal CA-630, and nucleases. The resulting matrices were allocated to: group I (control, n=10), crosslinked in 0.2% polymeric GA; and group II (treatment, n=10), crosslinked in 0.05% monomeric GA + ADAPT. Cytotoxicity was determined by in vitro cell seeding with human fibroblasts, and quantified using an Alamar Blue assay. The matrices were then implanted in a subcutaneous rat model and retrieved after extended implantation times (26 and 52 weeks). This was followed by further histology, immunohistochemical staining, and measurement of calcium deposition. Complete acellularity and biostability were significantly (p < 0.01) achieved in group II. Inflammatory responses were reduced and beneficial host cell infiltration with neocapillary formation was limited to group II. Fibroblast infiltration was significantly increased from six to 12 months' implantation time. Only group II tested positive for Factor VIII and vimentin. After 52 weeks, extractable calcium levels were significantly (p < 0.001) reduced in group II (2.56 +/- 0.22 microg Ca/mg tissue) compared to group I (136.02 +/- 0.39 microg Ca/mg tissue). Acellular bovine pericardium, when crosslinked with a low concentration of GA and treated with ADAPT, retains and improves its integrity with a low immunoreactivity over a prolonged period. Host cell infiltration is also optimized over time. The maintenance of reduced calcification levels in group II suggests that such a biomaterial might have an advanced long-term in vivo potential.