Anticalcification effects of decellularization, solvent, and detoxification treatment for genipin and glutaraldehyde fixation of bovine pericardium.

OBJECTIVE Calcification plays a major role in the failure of bioprosthetic and other tissue heart valve substitutes. The objective of this study was to evaluate the anticalcification effect of decellularization and detoxification in glutaraldehyde (GA)/genipin- and solvent-fixed bovine pericardium using a rabbit intramuscular model which is effective for assessing calcification in bioprosthetic tissue. METHODS Bovine pericardial tissues were fixed with 0.5% GA/0.4% genipin in organic solvent (75% ethanol + 5% octanol, 75% ethanol + 5% octanediol, or 70% ethanol + 10% isopropanol) and post-treated with glycine, glutamic acid, or sodium bisulfite. Decellularization was performed with 0.25% sodium dodecylsulfate prior to fixation. The material characteristics of the treated tissues were assessed by thermal stability test, uniaxial mechanical test, and light microscopy. Stability of the treated tissue was measured by the resistance to enzymatic degradation using protease. The tissues were intramuscularly implanted into 4-week-old rabbits for 8 weeks, and the anti-α-Gal(Gal[alpha]1,3-Gal[beta]1,4GlcNAc-R) antibodies (immunoglobulin G) were measured at various time intervals after implantation. Explanted tissues were examined by light microscopy and calcium contents of the explanted tissues were measured. RESULTS Differently treated tissues resulted in no significant alterations in material characteristics and morphology. GA groups are superior to genipin groups in tissue cross-linking without difference according to addition of decellularization, organic solvent treatment, and detoxification by resistance toward pronase degradation. The titer of anti-α-Gal antibodies gradually increased after implantation in all study groups. The titer of anti-α-Gal antibodies increased less in genipin groups than in GA groups, and less with decellularization than without decellularization. The calcium contents of genipin groups (n = 75) were significantly lower than those of GA groups (n = 118) (28.55 ± 11.22 μg mg(-1) vs 37.16 ± 7.75 μg mg(-1), p < 0.001). Calcium contents decreased with decellularization, organic solvent treatment, and detoxification, irrespective of the type of organic solvent and amino acids. Inorganic phosphorus contents of genipin groups (n = 66) were significantly lower than those of GA groups (n = 103) (55.36 ± 26.98 μg mg(-1) vs 75.67 ± 23.44 μg mg(-1), p = 0.000). CONCLUSIONS Genipin fixation is a novel alternative to conventional GA fixation in vitro material assessment and in vivo anticalcification effect. The addition of decellularization, organic solvent treatment, and detoxification prevented calcification of GA/genipin-fixed bovine pericardium in the rabbit intramuscular implantation model, irrespective of the type of organic solvent and amino acids.