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Objectives: Autologous pericardium rapidly fixed with glutaraldehyde (GA) in theatre is considered in many cardiac surgery centres the best material currently available for intracardiac, valvular or vascular repair. Implanted non-fixed autologous tissues suffer rapid degeneration, shrinkage and absorption whereas standard xenotypic fixed tissues cause local cytotoxicity and calcification. In the present study, using a subcutaneous rat model, we tested the biostability, durability and calcification potential of four different pericardium patches treated with GA and relevant to current clinical practice., Methods: Pericardium samples were divided into four groups according to the method of treatment. Group I consisted of bovine pericardium (BP) fixed with 0.6% GA (control), Group II cryopreserved human pericardium (CHP) rapidly fixed with 0.6% GA for 4 min and detoxified with MgCl2, Group III CHP treated with the multistep ADAPT(®) process (delipidized, decellularized with Tx-100, deoxycholate, IgePal CA-630 and denucleased, fixed in 0.05% monomeric GA and detoxified) and Group IV BP treated with the multistep ADAPT(®) process (CardioCel(®)). Biostability was determined by shrinkage temperature which measures the degree of cross-linking, and durability assessed by resistance to a mixture of proteinases (pronase digestion). Treated pericardium samples (n = 10 in each of Groups I-IV) were implanted in the subcutaneous rat model for 8 and 16 weeks, followed by histology and calcium analysis (atomic absorption spectrophotometry)., Results: The biostability and the durability of both CHP and BP after the multistep ADAPT(®) treatment remained stable without any microscopic calcification. Extractable calcium levels of CHP were significantly (P < 0.01) reduced in Group II (1.89 ± 0.77 μg Ca/mg tissue) compared with Group I (64.37 ± 6.25 μg/mg) after 8 weeks. Calcification of CHP (Group III) and BP (Group IV) after the multistep ADAPT(®) treatment was significantly reduced (1.43 ± 0.48 µg/mg and 0.75 ± 0.10 μg/mg, respectively) compared with Group I (282.52 ± 18.26 μg/mg) and the rapidly treated CHP in Group II (11.32 ± 3.21 μg/mg) after 16 weeks., Conclusions: Improved biostability and durability with reduced calcification of tissues after the multistep ADAPT(®) tissue treatment suggest improved alternative substitutes to autologous pericardium.

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 and Aim of the Study: Matrix preparation remains controversial due to incomplete cell removal, inflammatory responses, reabsorption and thrombocyte activation. Previously, crosslinked matrices have been considered unsatisfactory due to cytotoxicity. In the present study, the biostability, biocompatibility and calcification potential of a decellularized matrix crosslinked with a low concentration of monomeric glutaraldehyde (GA) and treated with the ADAPT anti-calcification process were examined., Methods: Bovine pericardium was decellularized with Triton X-100, deoxycholate, IgePal CA-630 and ribonuclease. The resulting matrices were allocated to either group I (control, n = 5), crosslinked in 0.2% polymeric GA + ADAPT, or to group II (treatment, n = 5), crosslinked in 0.05% monomeric GA + ADAPT. The physical properties, enzymatic degradation, histology and immunohistochemical staining of the tissues were monitored. The matrices were also implanted in the jugular vein of juvenile sheep for 200 days., Results: Complete acellularity was achieved. Biostability was significantly (p <0.01) enhanced in group II, but inflammatory responses were limited in both groups. Host fibroblasts infiltrated the periphery in group I and the entire matrix in group II. The luminal surfaces were free from thrombotic depositions and covered with endothelial cells. Both groups tested positive for Factor VIII, smooth muscle alpha-actin and vimentin. Tissue extractable calcium levels were low (group I = 1.02 +/- 0.39, group II = 0.86 +/- 0.22 microg Ca/mg tissue)., Conclusion: Low-concentration GA-crosslinked matrices proved to be stable. The immunoreactivity of both groups was low, with host cell infiltration, migration and trans-differentiation being optimized in those grafts crosslinked with an ultra-low monomeric GA concentration. Calcification levels were close to zero in both groups. Enhanced crosslinking and effective anti-calcification produce a biomaterial with advanced in-vivo tissue-engineering properties.

Aim: Bioprosthetic cardiovascular substitutes, manufactured from glutaraldehyde-preserved bovine or porcine tissues, are prone to calcification after implantation. The aim of the study was to evaluate the ultrastructure, material stability and calcification behaviour of glutaraldehyde-preserved bovine pericardium, treated with a multi-step anti-calcification process which addresses each of the major causes of calcification and tissue degeneration., Methods: Bovine pericardium samples were divided into 2 groups. Group I (control) consisted of tissue fixed with 0.625% glutaraldehyde and Group II (study group) consisted of tissue fixed with 0.625% glutaraldehyde and exposed to a multi-step anti-calcification process. Ultrastructure was examined by scanning electron microscopy and material stability was assessed by mechanical testing, shrinkage temperature and enzymatic degradation. Calcification was assessed by histology (Von Kossa stain) and by atomic absorption spectrophotometry in the subcutaneous rat model., Results: Bovine pericardium in the study group revealed less visible changes in the ultrastructure of the collagen matrix, improved material stability (P<0.05) and significantly (P<0.001) reduced calcification compared to control tissues (4.5+/-1.2 versus 136.03+/-11.39 ug/mg tissue)., Conclusions: In conclusion, results demonstrate that the multi-step anticalcification process improved the material stability and reduced the calcification potential of bovine pericardial tissue. These improvements in the quality of the bovine pericardium should enhance the long-term durability of the tissue as a bioprosthetic substitute for cardiovascular application.