title: Glutaraldehyde detoxification in addition to enhanced amine cross-linking dramatically reduces bioprosthetic tissue calcification in the rat model.
abstract: OBJECTIVE
  Enhanced fixation of bioprosthetic tissue by both increased concentrations of glutaraldehyde (GA) and the introduction of additional cross-links with L-lysine significantly reduces calcification. We have previously reported that prolonged exposure to high-volume amino-compounds under warm, acidic conditions leads to thorough, non-rebounding GA detoxification. The aim of the present study was to prove that removal of excess GA can amplify the benefits of enhanced GA cross-linking with regard to bioprosthetic tissue calcification.
  
  
  METHODS
  Porcine ascending aortas and leaflet tissue, and bovine pericardium were immediately fixed using three GA concentrations (0.2%, 1.0%, 3.0% (v/v)) for seven days at 4 degrees C. Samples were allocated to nine groups. Groups I to III received no further treatment (one at each GA concentration); groups IV to IX underwent an additional L-lysine interim step (48 h/37 degrees C/0.1 M) two days before completion of standard seven-day GA fixation; and groups VII to IX were additionally treated with a GA extraction process using high-volume urazole solution (acetic acid buffer, pH 4.5, 37 degrees C, one week) followed by NaBH4 reduction (2 days, 37 degrees C). Samples were implanted subcutaneously in rats (six per group) for six weeks. Tissue calcium was measured by atomic absorption spectrophotometry and examined histologically after von Kossa staining.
  
  
  RESULTS
  Calcification was reduced in all three tissue types by enhanced cross-linking and by extraction of excess GA. Increasing the GA concentration from 0.2% to 3.0% led to a reduction in calcification of 11.5% (p = 0.074; Student's t-test) in leaflets; 63.6% (p <0.0001) in pericardium; and 17.5% (p = 0.034) in aortic wall tissue. The introduction of additional cross-links with L-lysine resulted in a significant reduction of calcium in all tissues (maximally 42.5%, p = 0.0003 in leaflets; 79.3%, p = 0.005 in pericardium; and 49.6%, p <0.0001 in aortic wall; Student's t-test). Optimal reduction in calcification could be achieved with the combined effect of 3.0% GA fixation, L-lysine enhancement and urazole detoxification. When compared with 0.2% GA-fixed tissue, calcification could be reduced by 99.1% in leaflets, 95.9% in pericardium, and 90.8% in aortic wall tissue (p <0.0001 for all tissue types; Student's t-test).
  
  
  CONCLUSIONS
  Removal of excess GA from fixed bioprosthetic tissue was capable of markedly improving the anti-calcific effect of enhanced GA cross-linking.
authors.affiliation: Department of Cardiothoracic Surgery, Cape Heart Centre, University of Cape Town Medical School, South Africa.
authors.forename: C
authors.initials: C
authors.lastname: Weissenstein
authors.forename: P
authors.initials: P
authors.lastname: Human
authors.forename: D
authors.initials: D
authors.lastname: Bezuidenhout
authors.forename: P
authors.initials: P
authors.lastname: Zilla
mesh_terms: D000818:Animals; D001705:Bioprosthesis; D002114:Calcinosis / Q000473:pathology*; D002417:Cattle; D003432:Cross-Linking Reagents; D019544:Equipment Failure Analysis; D005404:Fixatives / Q000633:toxicity*; D005976:Glutaral / Q000633:toxicity*; D006350:Heart Valve Prosthesis; D008239:Lysine; D009926:Organ Preservation; D011183:Postoperative Complications / Q000473:pathology*; D051381:Rats; D020318:Rats, Long-Evans; D013552:Swine; D016707:Tissue Fixation
chemical_list: D003432:Cross-Linking Reagents; D005404:Fixatives; D008239:Lysine; D005976:Glutaral