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Background and Aims of the Study: 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., Methods: 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., Results: 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)., Conclusion: 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.

Background: Glutaraldehyde-preserved bioprosthetic heart valve substitutes have limited performance and longevity due to tissue degeneration and calcification. The Freestyle valve (Medtronic Heart Valves, Inc, Minneapolis, MN) combines zero fixation pressure and proportional, variant-amino oleic acid (AOA) as antidegeneration and antimineralization measures. The aim of this study was to compare the calcification behavior of glutaraldehyde-preserved kangaroo aortic valves with Freestyle stentless bioprostheses in a juvenile sheep model., Methods: Pulmonary artery and valve replacements were performed in juvenile sheep with Freestyle stentless aortic valves (n = 4) or glutaraldehyde-preserved kangaroo stentless aortic valves with no antimineralization measures (n = 6), and explanted at 200 postoperative days., Results: Freestyle stentless valves and stentless kangaroo aortic valves showed normal valve function immediately postoperatively and up to 120 days. Valve leaflets of all valves were macroscopically free of visible calcification with normal histology. Valve leaflet calcification (microg Ca/mg tissue) was less in kangaroo than Freestyle (1.27 +/- 0.43 versus 2.38 +/- 1.37, p = 0.856). Aortic wall tissue calcification was severe in kangaroo and Freestyle (127.93 +/- 12.22 versus 122.19 +/- 11.99, p = 0.596)., Conclusions: We conclude that glutaraldehyde-preserved kangaroo aortic valve leaflets are equal to AOA-treated Freestyle stentless valve leaflets with regard to calcification in juvenile sheep. Both bioprostheses are prone to aortic wall calcification. The low calcification features of the kangaroo aortic valve leaflets without antimineralization treatment may benefit the longevity of the valve.

Background and Aim of the Study: The study aim was to compare the crosslink stability and calcification behavior of porcine tissue (cusp and wall), treated with a new antimineralization process (ADAPT) with that of commercially prepared Freestyle and Prima Plus bioprosthetic tissues., Methods: Porcine aortic roots were divided into two groups: (i) tissue zero pressure-fixed with 0.625% glutaraldehyde (GA) for seven days and stored in 0.25% GA (as control); and (ii) tissue exposed to the ADAPT process for four days and stored in 0.25% buffered GA. These groups were compared with Freestyle and Prima Plus tissues (cusp and wall). Crosslink stability was assessed by shrinkage temperature and resistance to pronase degradation. Calcification behavior was assessed histologically (Von Kossa staining) and by atomic absorption spectrophotometry of explanted tissue after eight weeks. in a subcutaneous rat model., Results: Crosslink stability and calcification potential of ADAPT-treated porcine valve cusps were comparable to those of Freestyle and Prima Plus cusps (p = NS). ADAPT-treated porcine wall tissue showed improved crosslink stability (p <0.05) and significantly (p <0.001) reduced calcification (-95.95%) compared to control (-0.00%), Freestyle (-47.87%) and Prima Plus (-51.95%) tissues., Conclusion: The ADAPT process is effective in reducing calcification in both porcine cusp and wall tissues in a subcutaneous rat model, and further suggest that enhanced crosslinking plays an important role in minimizing aortic wall calcification.

Background and Aim of the Study: The performance and longevity of bioprosthetic heart valves are limited by tissue calcification and degeneration after implantation. Experimental valve replacement in large animal models forms an important component of the preclinical evaluation of these bioprosthetic heart valves. The study aim was to assess the feasibility of a mitral model for stentless valves and to evaluate the calcification behavior of stentless glutaraldehyde-preserved kangaroo heart valves in the mitral position of a sheep model., Methods: Medtronic Freestyle (n = 10) and kangaroo (n = 11) stentless aortic valves were implanted in the mitral position of juvenile sheep and retrieved after a maximum of 200 days. Retrieved stentless valves were examined for morphological changes and calcification of the valve tissue, using radiological screening, Von Kossa's staining and atomic absorption spectrophotometry., Results: Four sheep (40.0%) with Medtronic Freestyle and 10 sheep (90.9%) with kangaroo valves could be weaned from bypass and mechanical ventilation. Two animals (20.0%) with Medtronic Freestyle and six animals (54.5%) with kangaroo prostheses survived more than 30 days postoperatively. No significant difference (p >0.05) was seen between the calcification potential of Medtronic Freestyle valve leaflets (3.21 +/- 1.67 microg/mg) after 93 days and kangaroo valve leaflets (2.39 +/- 0.80 microg/mg) after 200 days., Conclusion: The present results suggest that implantation of a stentless valve in the mitral position of sheep is possible, but technically difficult. The calcification potential of kangaroo valve tissue is comparable to that of Freestyle valve tissue in the mitral position of sheep.

Stentless aortic heart valve substitutes, manufactured from biological tissues, are fixed with glutaraldehyde to cross-link collagen, reduce antigenicity, and sterilize the tissue. Despite improved cross linking, reduced antigenicity, and various anticalcification measures, the aortic wall tissue present in these prostheses tends to calcify. The aim of this study was to assess the morphology, collagen cross-link stability, and calcification potential of glutaraldehyde-preserved kangaroo aortic wall tissue as opposed to porcine aortic wall tissue. Porcine and kangaroo aortic wall tissues were fixed in 0.625% buffered glutaraldehyde. Histology and cross-link stability were examined. Calcification potential was determined in the subcutaneous rat model. Kangaroo aortic wall tissue was significantly (p < 0.01) less calcified than porcine aortic wall tissue (26.67 +/- 6.53 versus 41.959 +/- 2.75 microg/mg tissue) at 8 weeks. In conclusion, the histological differences between kangaroo and porcine aortic wall tissue correlate well with the reduced calcification potential of kangaroo aortic wall tissue. The reduced calcification potential could result in improved long-term durability of stentless kangaroo heart valves as bioprostheses., (Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 66B: 356-363, 2003)