Impact of processing methods on bovine pericardial tissue integrity
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Date
2020-01
Authors
Botes, Lezelle
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Publisher
University of the Free State
Abstract
Introduction: The use of cardiac patches remains one of the main therapeutic solutions for surgical treatment. Cardiovascular patches are either synthetic or biological. Synthetic materials have become less popular over the years because they are rigid, have poor flexibility, are surgically difficult to handle, are prone to endocarditis and local inflammatory reactions that contributes to fibrosis and calcification, and have no regeneration potential. Autologous pericardium tends to retract, thicken, become aneurysmal and develop fibrosis once implanted. Therefore, xenogeneic transplanted tissue dominated by bovine pericardium has become an attractive alternative. Glutaraldehyde (GA)-fixation was introduced to overcome the aggressive recipient graft-specific rejection response, ensure sterility and to increase durability and mechanical stability. However, the residual GA toxicity and host immune responses seen in GA-preserved bovine pericardium causes degenerative processes that involves structural changes causing rigidity, shrinkage, calcium deposition and subsequent failure of the pericardial patch. Furthermore, GA limits host cell infiltration, remodeling and fails to remove or mask all animal specific antigens that contributes to chronic rejection. This resulted in several strategies to reduce the side-effects of GA-fixation and to provide alternatives to GA as a crosslinking agent but with mixed results. Therefore, nowadays basic research is focused to produce a scaffold with reduced antigenicity while maintaining structural integrity and create recellularization potential. Attempts to reduce antigenicity included decellularization (e.g., sodium dodecyl sulphate (SDS), Triton X-100 (TX), trypsin), enzymatic or gene knockout removal of epitopes, and solubilization-based antigen removal. The Frater Cardiovascular Research Centre developed a proprietary decellularization protocol. The aim of the study was to evaluate the potential application of this technology by comparing the structural and morphological performance of decellularized scaffolds with and without GA-fixation and detoxification with two (2) commercially available patches, the Glycar® and Cardiocel® bovine pericardial patches after being implanted in a juvenile ovine model for 180-days. Methodology: A prospective analytical cohort design was followed. Four (4) groups of bovine pericardial patches were evaluated in vitro and in vivo namely; (i) Glycar® bovine pericardial patches (GA-fixated and detoxified), (ii) CardioCel® bovine pericardial patches (decellularized, GA-fixed and detoxified), (iii) a proprietary decellularized bovine pericardial scaffold (BPS) and (iv) a proprietary decellularized GA-fixed and detoxified (D-GAD) bovine pericardial patch. The patches/scaffolds of each group were implanted in the descending aorta and main pulmonary artery of six (6) juvenile whether sheep per group for a minimum of 180-days. The clinical and mechanical integrity, tissue morphology and pericardial thickness were evaluated and compared between the four (4) bovine pericardial groups prior to implantation and after explantation. Results: The impact of glutaraldehyde-fixation and detoxification (GAD) technology on tissue seems to be constant, irrespective whether the tissue was decellularized or not. The Glycar®, CardioCel® and D-GAD tissues handled well, had excellent clinical outcomes and did not calcify. The tensile strength (TS) decreased from preimplantation to explantation in all three (3) groups but still exceeded the strength of the native human aorta (1.8 ± 0.24 MPa). The pericardial tissue was more pliable in all three groups after explantation compared to their pre-implantation counterparts. A fibrous encapsulation developed on all these explants, explaining the thickening of the pericardial patches. The collagen of the GAD groups was compact and dense which reduces the pore sizes to promote host cell infiltration. Host cell infiltration of fibroblast-like cells was insignificant in the explanted aorta and pulmonary Glycar® patches and limited in the CardioCel® and D-GAD patches. None of the patches in the three (3) groups remodelled after implantation. Only the Glycar® patches demonstrated endothelial cells prior to implantation although severely dehydrated. However, at explantation all three (3) groups demonstrated a monolayer of endothelial cells on the pericardial surface. The fibrous encapsulation caused thickening of both the aortic and pulmonary patches in the Glycar® and CardioCel® groups but in the D-GAD group only the pulmonary patch increased in thickness. The surgical handling of the decellularized BPS was satisfactory however, surgeons did comment that it was slippery. Furthermore, it had excellent clinical outcomes and did not calcify, disintegrate or developed aneurysms. The TS decreased from pre implantation to explantation but still exceeded the strength of the native human aorta (1.8 ± 0.24 MPa). The pericardial tissue was more pliable at explantation compared to its pre-implantation counterpart. No fibrous encapsulation developed, and the pericardial patches did not thicken over time. The collagen of the BGS groups was wavy and well-separated providing large pore sizes to promote host cell infiltration. Significant host cell infiltration of fibroblast-like cells was demonstrated in both the explanted aorta and pulmonary scaffolds. The scaffold completely remodelled after implantation. Endothelial cells were absent prior to implantation but at explantation a monolayer of endothelial cells were visible on the pericardial surface. Conclusion: All four (4) pericardial groups demonstrated excellent clinical, structural and morphological results when implanted in a juvenile ovine model for 180-days. Adding decellularization to the processing process benefits the collagen matrix, by making the collagen less dense/compact thus allowing for larger pore sizes. This may allow for better recellularization, especially in the absence of cells or cellular debris, invoking an immunological reaction. The long-term benefit of the limited recellularization seen in both D-GAD products remains to be clearly demonstrated. However, the decellularized BPS should be considered as an alternative to GA-fixed pericardial patches for it demonstrated excellent remodeling and growth potential. The importance of remodelling may be beneficial to the long-term outcomes especially in the younger age group recipients.
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Keywords
Thesis (Ph.D. (Cardiothoracic Surgery))--University of the Free State, 2020, Bovine - Pericardial tissue integrity., Bovine pericardium.