Original articleThrombogenicity studies of three different variants of processed bovine pericardiumÉtudes de thrombogénécité de trois différents variants des péricardes de bovins traités
Introduction
Bovine pericardium (BP) processing towards a clinical biomaterial is a common practice. Bovine pericardium tissue patches have been used for pericardial sac closure after open-heart surgery [2], vascular graft repair [1], [18] and in making a heart valve [12], [24].
It basically comprises of collagen type I, which has got low antigenicity, but high thrombogenicity [14]. It is prepared acellular and cross-linked by chemical or physical methods to reduce antigenicity [8], thereby preventing calcification this enhances the mechanical strength and minimizes xenogenic tissue solubility [3], [17]. Standard practice of glutaraldehyde cross-linking carries a high-risk of tissue fatigue and calcific degeneration [7], partly due to cytotoxicity and inflammatory changes [11], and fragmentation of collagen because of continuous wear and tear. To address the issue of calcification, Lee et al. developed a novel methodology for the chemical modification of biological tissues by directly coupling heparin to bovine pericardium (BP). Their heparinization involved pretreatment of BP using glutaraldehyde (GA) and was followed by grafting heparin to BP by the reaction of residual aldehyde with amine group of heparin. They evaluated the effect of heparin coupling on calcification by in vitro as well as in vivo studies. Their results revealed that heparinized BP exhibit greater resistance to collagenase digestion than control tissue [25].
The aim of this study was to find a better processing technique with effective cross-linking to produce a durable biomaterial, which remains flexible, biocompatible and nonthrombogenic. While analyzing the various methods for processing of the biomaterial, our focus was on thrombogenic studies. In this study, we made the tissue acellular, followed by cross-linking with heparin and subsequent treatment via anticalcification and then analysed for major thrombogenic parameters.
Section snippets
Materials and methods
Bovine pericardium was harvested and procured from an inspected abattoir in sterile environment. The specimens were stored in Hanks balanced salt solution (HBSS) having an antibiotic cocktail of cifran, gentamycin, streptomycin, cephalosporin and amphotericin B, and later brought into a current Good Manufacturing Practice (cGMP) laboratory for further processing.
Forty-nine bovine pericardial pieces were taken for study, where 20 numbers identified as group A processing were treated with
Results
All the groups had complete decellularisation by the standard method followed in the laboratory. High-resolution optical microscopy had shown better tissue homogeneity and integrity in group B which was supported by confocal microscopy (CM), and transmission electron microscopy (TEM). Higher tissue density was noticed by ESEM, in case of group B. Gamma irradiated tissue showed granular appearance by CM and TEM, fragmentation by ESEM, whereas group A ones showed nonintegrated separated matrix,
Discussion
Though bovine pericardium has potential to emerge as an excellent graft material, it has been plagued by problems inherent or due to inadequate processing techniques [7] (Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8). Decellularized bovine pericardium has propensity for calcification, unless the collagen is effectively cross-linked. Further calcification can induce thrombosis or vice versa [4] Cross-linking with heparin prevents early tissue deterioration by forming an artificial matrix made up of
Conclusion
Use of heparin as a cross-linking agent results in a superior biomaterial reflected by its favorable thrombogenic profile, mechanical and surface properties (smooth without pitting). Our study also indicates that gamma sterilization results in deleterious effect on the surface and mechanical properties of the tissue. It is to be noted that heparin cross-linking without gamma irradiation produces the best biomaterial and has been proved to be sterile. The validity of these results and
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A review of current approaches for decellularization, sterilization, and hemocompatibility testing on xenogeneic pericardium
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